Lipocalin fusion partners

ABSTRACT

Methods and systems for producing fusion proteins and peptides are disclosed. Fusion proteins and peptides created using the methods are also provided. Also provided are methods of using the fusion proteins and peptides produced according to the present disclosure.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/651,175, filed Jun. 10, 2015, which is a National Phase Applicationbased on International Patent Application No. PCT/US2013/074215, filedDec. 10, 2013, which claims priority to U.S. Provisional PatentApplication Nos. 61/735,516, filed Dec. 10, 2012 and 61/794,685, filedMar. 15, 2013, all of which are incorporated herein by reference intheir entirety as if fully set forth herein.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with the support of the United States governmentunder Contract numbers R01CA135491, NIH ROI A1059543, A1094419, andA1097786 by the National Institutes of Health.

BACKGROUND OF THE INVENTION

Efforts toward drug discovery continue to use vast technical andfinancial resources to identify and develop new and useful drugs.Unfortunately, finding new drugs has continued to be difficult. Forexample, development of less damaging, more precisely targeted cancertherapies is essential. But even after decades of research, scientistsstill struggle to identify therapeutic compounds with the right mix ofmedicinal and cancer-targeting properties which not only reduce thelikelihood that a therapeutic compound could serve as a treatmentstrategy but creates a need for successful methods of surgicalresection.

A wide variety of types of compounds have been studied and pursued for alarge breadth of therapeutic purposes. For example, small chemicalmolecules and larger biologics (e.g., antibodies) have been used for aplethora of therapeutic applications with varied success. Some smallerpeptides have also been shown to be useful as drugs, e.g., by virtue oftheir natural potency.

A lack of methods for rapid and efficient production of peptides andproteins for clinical applications has limited the discovery of peptidesand proteins that might serve as therapeutic compounds. Creating fusionsof peptides, protein domains, or proteins, to a different protein toenhance production of peptides and proteins has been explored inbacterial expression systems, e.g., E. coli gene expression. However,bacterial protein expression systems are generally limited due to errorsin protein folding efficiency. Thus, there is still a need for methodsenabling the rapid and efficient production of peptides, proteindomains, and proteins.

SUMMARY OF THE INVENTION

The present invention relates to methods for the production ofsiderocalin fusion proteins. In various aspects, the present inventionrelates to the fusion proteins produced according to those methods. Incertain apsects, the fusion protein is cleaved, thereby producing apeptide according to the present disclosure. The present inventionfurther relates to methods for producing a fusion protein, the methodscomprising expressing, in a cell, a fusion protein, the fusion proteincomprising a peptide or protein domain and a lipocalin protein, therebyproducing the fusion protein. In some embodiments, the present inventionfurther relates to a method of producing a peptide, the methodcomprising: expressing, in a cell, a fusion protein comprising a peptideand a lipocalin protein; and separating the peptide from the lipocalinprotein, thereby producing the peptide.

In some embodiments, the present invention relates to a composition of afusion protein, the composition comprising; a peptide or protein domain,and; a lipocalin protein. In some embodiments, the present inventionfurther relates to a composition comprising a peptide library, thepeptide library further comprising a plurality of peptides lacking atleast one native lysine residue. The present invention further relatesto a composition comprising a fusion protein comprising a peptide and alipocalin protein.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 provides a gel image showing a variety of scaffolds, inaccordance with an embodiment of the present invention.

FIG. 2 shows an example method of producing peptide libraries, inaccordance with an embodiment of the present invention.

FIG. 3 depicts an example fusion system that can be used to makeknottins (e.g., bubble protein), in accordance with an embodiment of thepresent invention.

FIG. 4 shows analysis of a large peptide library using massspectrometry, in accordance with an embodiment of the present invention.

FIG. 5 shows an example method of using siderocalin fusions to expressknottin variants, in accordance with an embodiment of the presentinvention.

FIG. 6 depicts SDS-PAGE analysis of expressing knottin scaffolds, inaccordance with embodiments of the present invention.

FIGS. 7A and 7B provide a schematic of pooled library production, inaccordance with an embodiment of the present invention. FIG. 7B shows anexpanded view of FIG. 7A.

FIG. 8 describes representative sequencing data from a cloned knottinlibrary, in accordance with an embodiment of the present invention.

FIG. 9 shows SDS-PAGE analysis of 3000-member knottin libraries, inaccordance with embodiments of the present invention.

FIG. 10 shows a generalized process for manufacturing knottins, inaccordance with an aspect of the present disclosure.

FIG. 11 shows a generalized process for manufacturing knottins, inaccordance with an aspect of the present disclosure.

FIG. 12 a generalized process for manufacturing knottins, in accordancewith an aspect of the present disclosure. According to this aspect,cleavage occurs at the furin-knottin interface.

FIG. 13 shows examples of knottins made with the Daedalus system andcorresponding SDS PAGE analyses in accordance with an aspect of thepresent disclosure. The Daedalus system is desribed in Bandaranayake A.D., et al., Nucleic Acids Res. (2011) 39(21):e143.

FIG. 14 shows the alignment of Scn sequences from 18 species. Thealignment shows a high level of sequence conservation between the 18species with recognizable orthologs. Positions with an asterisk arepossible sites for ligand binding. Correnti, C. & Strong, R. K. (2013)‘Iron sequestration in immunity’ In Metals in Cells; Encyclopedia ofInorganic and Bioinorganic Chemistry. (Culotta, V. & Scott, R. A., eds.)John Wley & Sons, pp. 349-59

FIG. 15 depicts a 3D model of super-stable Scn in accordance with anaspect of the present disclosure. In this form of Scn, a seconddisulfide bond was engineered in order to secure the N-terminus andincrease thermal stability.

FIG. 16 shows a schematic of a generic Scn fusion that can be used invarious aspects of the present disclosure. The native signal peptide wasbeen removed and an exogenous sFLAG and HIS tag were added to facilitatepurification. These modifications are optionally present in variousaspects of the disclosure.

FIG. 17 depicts a schematic of a light chain antibody fusion with Scnand corresponding SDS PAGE analysis according to an aspect of thepresent disclosure.

FIG. 18 shows the SDS PAGE analysis comparing the expression of a Scnfusion with a cytoplasmic enzyme, HMOX1, both before and after cleavageaccording to an aspect of the present dislcosure.

FIG. 19 depicts the expression of a Scn fusion with a cytoplasmic viralprotein Adv2 and the corresponding SDS PAGE analysis according to oneaspect of the present disclosure.

FIG. 20 depicts the expression of a Scn fusion with an extracellularviral glycoprotein HIV gp120 and the corresponding SDS PAGE analysisaccording to one aspect of the present disclosure.

FIG. 21 depicts the expression of a Scn fusion with a knottin protein,Imperatoxin, and the corresponding SDS PAGE analysis according to oneaspect of the present disclosure.

FIG. 22 depicts the expression of a Scn fusion with a small subdomain(i.e., Kringle domain) of the extracellular tyrosine kinase receptorROR1 and corresponding SDS PAGE analysis according to one aspect of thepresent disclosure.

FIG. 23 depicts the expression of a Scn fusion with heptameric andtrimeric subdomains and corresponding SDS PAGE analysis according to oneaspect of the present disclosure.

FIG. 24 depicts the expression of an ExFABP fusion with a knottin andcorresponding SDS PAGE analysis according to one aspect of the presentdisclosure. ExFABP is another functional Scn. According to the presentdisclosure, this construct can be used in a periplasmic bacterial systemto secret a variety of client proteins.

FIG. 25 depicts the crystal structure of Scn with a Th ligand.

FIG. 26 depicts a captured fluorescent siderophore. The left framedepicts a size-exclusion purification of the protein siderophorecomplex. The right frame is a schematic.

DETAILED DESCRIPTION OF THE INVENTION

The methods and systems of the present disclosure relate to fusionproteins and methods of producing them. According to certain aspects,the peptides produced using the present methods can be used ascomponents of drug discovery platforms. In some aspects, the methodsrelate to creating a fusion of a first protein, or of a peptide, thathas potential therapeutic value, with a second protein such that thesecond protein enhances production and folding of the first protein byan expression system. In some aspects, after expression and purificationof the fusion protein, the fusion protein is cleaved such that the firstprotein and the second protein are separate proteins. In some aspects,the second protein may be a lipocalin. For example, the second proteinmay be a specific lipocalin such as siderocalin.

Conjugates

In some aspects, the present invention includes fusion proteins,peptides, or conjugates thereof as described herein. For example, someor all of the fusion proteins or peptides can be conjugated to a moietyselected to modify a property of the peptides.

In certain aspects, the present invention includes fusion proteins orpeptides conjugated at the N-terminus to hydrophobic (e.g., lipophilic)moieties. All or some of the fusion proteins or peptides of the presentdisclosure can be lacking internal lysines, e.g., to avoid conjugationat the internal lysine positions, thereby allowing conjugation to theamino terminus of the peptide. In some embodiments, the attachment of ahydrophobic moiety to the N-terminus can be used to extend half-life ofthe fusion protein or peptide of the present disclosure. In someembodiments, simple carbon chains (e.g., by myristoylation and/orpalmitylation) can be conjugated to the fusion proteins or peptides. Insome aspects, the simple carbon chains may render the fusion proteins orpeptides easily separable from the unconjugated material. For example,methods that may be used to separate the fusion proteins or peptidesfrom the unconjugated material include, but are not limited to, solventextraction and reverse phase chromatography. The lipophilic moieties canextend half-life through reversible binding to serum albumin. In certainembodiments, attachment of a near infrared dye to the N-terminus of thefusion protein or peptide can also be performed to allow for tracing ofthe conjugated fusion protein or peptide. In certain embodiments,attachment of a near infrared dye to a lysine of the peptide can also beperformed to allow for tracing of the conjugated peptide. An antibody tothe dye can further allow the dye to fill a dual role of both a trackingmarker and a retrieval handle. The conjugated fusion proteins orpeptides can also be conjugated to other moieties that can serve otherroles, such as providing an affinity handle (e.g., biotin) for retrievalof the peptides from tissues or fluids.

Other modifications can be used. For example, the fusion proteins,peptides, or conjugates thereof can include post-translationalmodifications (e.g., methylation and/or amidation). In some embodiments,the fusion proteins or peptides of the present disclosure can beconjugated to other moieties that, e.g., can modify or effect changes tothe properties of the peptides. The conjugated moieties can, e.g., belipophilic moieties that extend half-life of the peptides throughreversible binding to serum albumin. In some embodiments, the lipophilicmoiety can be cholesterol or a cholesterol derivative includingcholestenes, cholestanes, cholestadienes and oxysterols. In someembodiments, the peptides can be conjugated to myristic acid(tetradecanoic acid) or a derivative thereof.

In some embodiments, the fusion proteins or peptides of the presentdisclosure can be conjugated to detectable labels to enable trackingdetecting or visualizing of the bio-distribution of a conjugatedpeptide. The detectable labels can be fluorescent labels (e.g.,fluorescent dyes). In certain embodiments, the fluorescent label canhave emission characteristics that are desired for a particularapplication. For example, the fluorescent label can be a fluorescent dyethat has a emission wavelength maximum between a range of 500 nm to 1100nm, between a range of 600 nm to 1000 nm, between a range of 600 to 800nm, between a range of 650 nm to 850 nm, between a range of 700 nm to800 nm, between a range of 720 to 780 nm, or between a range of 720 to750 nm. For example, under certain conditions, cyanine 5.5 can have anemission maximum around 695 nm, IRdye 800 can have an emission maximumaround 800 nm, and indocyanine green can have an emission maximum around820 nm. One of ordinary skill in the art will appreciate the variousdyes that can be used as detectable labels and that have the emissioncharacteristics above.

As used herein, the term “detectable label” means a tag or modificationthat can be attached to a small chemical molecule, peptide, protein, ora fragment or a portion thereof such that the small chemical molecule,peptide, protein, or a fragment thereof is recognizable using a device,apparatus or method that permits the detection of the tag ormodification.

In some aspects, the detectable label is a fluorescent dye. Non limitingexamples of fluorescent dyes that could be used as a conjugatingmolecule in the present disclosure include rhodamine, rhodol,fluorescein, thiofluorescein, aminofluorescein, carboxyfluorescein,chlorofluorescein, methylfluorescein, sulfofluorescein, aminorhodol,carboxyrhodol, chlororhodol, methylrhodol, sulforhodol; aminorhodamine,carboxyrhodamine, chlororhodamine, methylrhodamine, sulforhodamine, andthiorhodamine, cyanine, indocarbocyanine, oxacarbocyanine,thiacarbocyanine, merocyanine, a cyanine dye (e.g., cyanine 2, cyanine3, cyanine 3.5, cyanine 5, cyanine 5.5, cyanine 7), oxadiazolederivatives, pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, pyrenederivatives, cascade blue, oxazine derivatives, Nile red, Nile blue,cresyl violet, oxazine 170, acridine derivatives, proflavin, acridineorange, acridine yellow, arylmethine derivatives, xanthene dyes,sulfonated xanthenes dyes, Alexa Fluors (e.g., Alexa Fluor 594, AlexaFluor 633, Alexa Fluor 647, Alexa Fluor 700), auramine, crystal violet,malachite green, tetrapyrrole derivatives, porphyrin, phtalocyanine, andbilirubin. In some embodiments, the dyes can be near-infrared dyesincluding, e.g., Cy5.5, IRdye 800, DyLight 750 or indocyanine green(ICG). In some embodiments, near infrared dyes can include cyanine dyes(e.g., cyanine 2, cyanine 3, cyanine 3.5, cyanine 5, cyanine 5.5,cyanine 7). In certain embodiments, the detectable label can includexanthene dyes or sulfonated xanthenes dyes, such as Alexa Fluors (e.g.,Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 700). Ifan antibody to the dye could be found the conjugated dyes could be usedboth as a tracking, detecting or visualizing marker and as a retrievalhandle.

The fusion proteins or peptides of the present invention can also beconjugated to biotin. In addition of extension of half-life, biotincould also act as an affinity handle for retrieval of the peptides fromtissues or other locations. In one embodiment, the peptides can beconjugated, e.g., to a biotinidase resistant biotin with a PEG linker(e.g., NHS-dPEG₄-Biotinidase resistant biotin). In some embodiments,fluorescent biotin conjugates that can act both as a detectable labeland an affinity handle can be used. Non limiting examples ofcommercially available fluorescent biotin conjugates include Atto425-Biotin, Atto 488-Biotin, Atto 520-Biotin, Atto-550 Biotin, Atto565-Biotin, Atto 590-Biotin, Atto 610-Biotin, Atto 620-Biotin, Atto655-Biotin, Atto 680-Biotin, Atto 700-Biotin, Atto 725-Biotin, Atto740-Biotin, fluorescein biotin, biotin-4-fluorescein,biotin-(5-fluorescein) conjugate, and biotin-B-phycoerythrin, alexafluor 488 biocytin, alexa flour 546, alexa fluor 549, lucifer yellowcadaverine biotin-X, Lucifer yellow biocytin, Oregon green 488 biocytin,biotin-rhodamine and tetramethylrhodamine biocytin. In some otherexamples, the conjugates could include chemiluminescent compounds,colloidal metals, luminescent compounds, enzymes, radioisotopes, andparamagnetic labels.

In some aspects, the fusion proteins and peptides of the presentinvention can be conjugated to vitamins or other molecules typicallyfound in foods that are absorbed into the bloodstream from the stomach,small intestine, or colon. Examples include, but are not limited to,vitamin A, vitamin C, vitamin B2, vitamin B3, vitamin B6, vitamin B12,vitamin D, vitamin E, vitamin K. The goal of these conjugations is toimprove oral bioavailability or absorption of the peptide from thegastrointestinal system.

In some instances, selected series of amino acids that appear to helpcertain peptides cross biologic barriers such as the gastrointestinaltract, the blood brain barrier, the cell membrane, the nuclear membranecan be identified and genetically or physically grafted onto otherpeptides for the purpose of helping the new peptide cross the samebiologic barriers. In other cases, the same approach might be used tograft sequences onto peptides that would prevent the new peptide fromcrossing certain biological barriers. For example, a drug could bemodified in this manner to prevent BBB penetration and thus reduce thelikelihood of central nervous system side effects.

In certain embodiments, the fluorescent label can have emissioncharacteristics that are desired for a particular application. Forexample, the fluorescent label can be a fluorescent dye that has aemission wavelength maximum between a range of 500 nm to 1100 nm,between a range of 600 nm to 1000 nm, between a range of 600 to 800 nm,between a range of 650 nm to 850 nm, between a range of 700 nm to 800nm, between a range of 720 to 780 nm, or between a range of 720 to 750nm. One of ordinary skill in the art will appreciate the various dyesthat can be used as detectable labels and that have the emissioncharacteristics above. For example, under certain conditions, cyanine5.5 can have an emission maximum around 695 nm, IRdye can have anemission maximum around 800 nm, and indocyanine green can have anemission maximum around 820 nm.

Non-limiting examples of fluorescent dyes that could be used as aconjugating molecule in the present disclosure include rhodamine,rhodol, fluorescein, thiofluorescein, am inofluorescein,carboxyfluorescein, chlorofluorescein, methylfluorescein,sulfofluorescein, aminorhodol, carboxyrhodol, chlororhodol,methylrhodol, sulforhodol; aminorhodamine, carboxyrhodamine,chlororhodamine, methylrhodamine, sulforhodamine, and thiorhodamine,cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine,merocyanine, oxadiazole derivatives, pyridyloxazole,nitrobenzoxadiazole, benzoxadiazole, pyrene derivatives, cascade blue,oxazine derivatives, Nile red, Nile blue, cresyl violet, oxazine 170,acridine derivatives, proflavin, acridine orange, acridine yellow,arylmethine derivatives, auramine, crystal violet, malachite green,tetrapyrrole derivatives, porphyrin, phtalocyanine, and bilirubin. Insome embodiments, the detectable label can include near-infrared dyes,such as, but not limited to, Cy5.5, indocyanine green (ICG), DyLight 750or IRdye 800. In some embodiments, near infrared dyes can include acyanine dye (e.g., cyanine 2, cyanine 3, cyanine 3.5, cyanine 5, cyanine5.5, cyanine 7). In certain embodiments, the detectable label caninclude xanthene dyes or sulfonated xanthenes dyes, such as Alexa Fluors(e.g., Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor700). In addition, if an antibody to the dyes can be identified, thenconjugated dyes could be used both as a tracking, detecting orvisualizing marker and as a retrieval handle.

Other modifications to fusion proteins, peptides, or conjugates thereofof the present disclosure can be used. For example, the fusion proteinsor peptides of the present disclosure can include post-translationalmodifications (e.g., methylation and/or amidation), which can affect,e.g., serum half-life. In some embodiments, the fusion proteins orpeptides can be conjugated to other moieties that, e.g., can modify oreffect changes to the properties of the peptides. The conjugatedmoieties can, e.g., be lipophilic moieties that extend half-life of thepeptides through reversible binding to serum albumin. In someembodiments, simple carbon chains (e.g., by myristoylation) can beconjugated to the peptides. In some embodiments, the lipophilic moietycan be cholesterol or a cholesterol derivative including cholestenes,cholestanes, cholestadienes and oxysterols. In some embodiments, thepeptides can be conjugated to myristic acid (tetradecanoic acid) or aderivative thereof.

The fusion proteins or peptides of the present disclosure can also beconjugated to other moieties that can serve other roles, such asproviding an affinity handle (e.g., biotin) for retrieval of thepeptides from tissues or fluids. For example, the peptides of thepresent invention can also be conjugated to biotin. In addition toextension of half-life, biotin could also act as an affinity handle forretrieval of the peptides from tissues or other locations. In someembodiments, fluorescent biotin conjugates that can act both as adetectable label and an affinity handle can be used. Non limitingexamples of commercially available fluorescent biotin conjugates includeAtto 425-Biotin, Atto 488-Biotin, Atto 520-Biotin, Atto-550 Biotin, Atto565-Biotin, Atto 590-Biotin, Atto 610-Biotin, Atto 620-Biotin, Atto655-Biotin, Atto 680-Biotin, Atto 700-Biotin, Atto 725-Biotin, Atto740-Biotin, fluorescein biotin, biotin-4-fluorescein,biotin-(5-fluorescein) conjugate, and biotin-B-phycoerythrin, Alexafluor 488 biocytin, Alexa flour 546, Alexa Fluor 549, lucifer yellowcadaverine biotin-X, Lucifer yellow biocytin, Oregon green 488 biocytin,biotin-rhodamine and tetramethylrhodamine biocytin. In some otherexamples, the conjugates could include chemiluminescent compounds,colloidal metals, luminescent compounds, enzymes, radioisotopes, andparamagnetic labels.

Methods of Making Fusion Proteins and Peptides

In yet another aspect, the present invention includes methods for makingfusion proteins or peptides according to the present disclosure.

In some embodiments, the present invention includes methods of makingfusion proteins or peptides according to the present disclosure. Asdescribed further herein, the present invention includes scaffolds thatcan be used as a starting point for generating fusion proteins orpeptides according to the present disclosure. These scaffolds as well asa large diversity of scaffold variants can be made using severaldifferent approaches. In some aspects, the fusion proteins or peptidesaccording to the present disclosure can be produced using peptidesynthesis techniques generally well known in the art. Conventionaloligonucleotide synthesis techniques (e.g., chip-based oligonucleotidesynthesis) can also be used. In some instances, the synthetic approachescan be combined with a variety of expression systems. In one exampleembodiment, particular residue positions in a scaffold can be targetedfor random mutagenesis using degenerate codons to generate a diverse setof DNAs that can be made using, e.g., chip-based oligonucleotidesynthesis and can code for a large library of scaffold variants.

In some embodiments, the molecules coding for the scaffolds and scaffoldvariants can be expressed in various expression systems, and can, insome embodiments, be combined as part of a fusion system. The DNAmolecules encoding the scaffolds and scaffold variants, e.g., can becombined with fusion systems that can be expressed in several differentcell types, e.g., 293 HEK or E. coli. Fusions for 293 HEK cells, e.g.,can include but are not limited to, IgK leader sequences and/or secretedfusion proteins, such as siderocalin, lipocalin 2, and human serumalbumin.

In some embodiments, the peptides described herein (e.g., knottedpeptides) can be expressed as fusions with lipocalin proteins. In oneaspect, the present invention includes a method for producing a peptidethat can include expressing, in a cell, a fusion protein including apeptide (e.g., a knotted peptide) and a lipocalin protein. The methodcan further include separating the peptide from the lipocalin protein,thereby producing the peptide (e.g., the knotted peptide). The presentinvention further includes compositions of the fusion protein includingthe lipocalin protein and the peptide (e.g., the knotted peptide). Thisfusion system offers a variety of advantages for producing peptides(e.g., knotted-peptides) over traditional fusion systems. By way ofbackground, and not to be limiting in any way, the lipocalins are aclass of proteins that can have a conserved fold characterized by aneight-stranded beta barrel with a flanking alpha helix. The expressionlevels of lipocalin proteins, like Lcn2, NGAL and Siderocalin, inmammalian cells equal or surpass many other fusion systems, including Fcfusions. The present invention relates to methods for producing a fusionprotein, the methods comprising expressing, in a cell, a fusion protein,the fusion protein comprising a peptide or protein domain and alipocalin protein, thereby producing the fusion protein. In someembodiments, the methods further comprise separating the peptide orprotein domain from the lipocalin protein, thereby producing a peptideor protein domain. In certain embodiments, the peptide or protein domainis an antibody fragment. In certain embodiments, the antibody istrastuzumab, infliximab, adalimumab, OKT3, or Fc.

In some embodiments, the peptide or protein domain is human hemeoxygenase 1 or murine heme oxygenase 1. In some embodiments, the fusionprotein further comprises a cleavage site. In certain embodiments, thecleavage site is a furin cleavage site, a trypsin cleavage site or a TEVcleavage site. In some embodiments, the separating of the peptide orprotein domain from the lipocalin protein results from cleavage at thecleavage site in the fusion protein. In certain embodiments, theseparating of the peptide or protein domain from the lipocalin proteinoccurs following secretion of the fusion protein from a cell. In certainembodiments, the cell is a mammalian cell.

The present invention relates to methods of producing a peptide, themethod comprising: expressing, in a cell, a fusion protein comprising apeptide and a lipocalin protein; and separating the peptide from thelipocalin protein, thereby producing the peptide. In some embodiments,the peptide is separated from the lipocalin protein by proteolysis or bycleavage of a furin cleavage site in the fusion protein such that thepeptide is cleaved from the lipocalin protein upon secretion from thecell. In certain embodiments, the cell is a mammalian cell. In someembodiments, the peptide is produced at a concentration less than about200 mg/liter.

In some embodiments, the present invention relates to compositionscomprising a fusion protein comprising a peptide and a lipocalinprotein. In some embodiments, the peptide comprises a knotted peptide.In some embodiments, the knotted-peptide is selected from the groupconsisting of chymotrypsin inhibitor, hefutoxin, bubble protein, theC-terminal domain of midkine, potato carboxypeptidase inhibitor, andepiregulin. In certain embodiments, the lipocalin protein issiderocalin. In certain embodiments, the knotted-peptide includes atleast two disulfide bonds

The peptides described herein (e.g., knotted peptides) can be expressedusing a variety of lipocalin proteins. As used herein, the term“lipocalin” refers to a protein as defined in “Structure and sequencerelationships in the lipocalins and related proteins”, Darren R. Flower,Anthony C. T. North, Teresa K. Attwood, Protein Science (1993) 2:5,753-761. Lipocalins may include, but are not limited to, the numberedlipocalins (e.g., Lcn2 (also NGAL, Siderocalin, 24p3), and the like,chicken Ex-FABP and quail Q86.

In various apsects of the present disclosure, siderocalin is used as asecretion partner. As used herein, the term “siderocalin” refers to alipocalin that is capable of binding a small chelator. In some aspects,a chelator may be natural or engineered. In some aspects, siderocalinbinds to siderophores and ferric siderophore complexes. For example, asiderocalin can be, but is not limited to, a siderocalin or Ex-FABP. Asused herein, the term “Siderocalin,” (as a proper noun), refers to theorthologous family of proteins related to the human archetypeSiderocalin.

Siderocalin advantageously can be used for the secretion of a variety ofpeptides, proteins, and protein domains, including intracellularpeptides, proteins, and peptide domains when used as a secretionpartner.

Siderocalin is useful as a fusion partner for larger proteins because,e.g., of the small size of siderocalin relative to larger proteins (themature protein is 178 amino acids and has a molecular weight of 20547Da). Also, a C87S mutation in siderocalin can prevent dimerization andyields pure monomeric fusion protein (see Goetz, D. H., et al. ‘TheNeutrophil Lipocalin NGAL is a Bacteriostatic Agent that Interferes withSiderophore-mediated Iron Acquisition’ Molecular Cell (2002) 10:1033-43). A single intramolecular disulfide bond present in siderocalinincreases its stability. Also, siderocalin only has a single N-linkedglycosylation site, which involves correct processing in the ER beforesecretion. In some aspects, the peptides can also be expressed as fusionpeptides with Murine SCN (also known as 24p3), which also works verywell as a secretion partner. Other homologs can also be used. Inaddition, the peptides (e.g., knotted peptides) provided herein can alsobe expressed as fusion systems with the other members of the lipocalinfamily including Lcn1, Lcn6, Lcn8, Lcn9, Lcn10, Lcn12, Lcn15. In someembodiments, the peptide comprises a disulfide knotted-peptide. Incertain embodiments, the knotted-peptide is PMP-D2, potatocarboxypeptidase, huwentoxin, imperatoxin, epiregulin, midkine, bubbleprotein or conotoxin CVIC. In other embodiments, the peptide comprises aknottin. In some embodiments, the lipocalin protein comprisessiderocalin. In some embodiments, the lipocalin protein is siderocalin.In certain embodiments, the lipocalin protein is human siderocalin,murine siderocalin, chicken Ex-FABP, or quail Q86.

In some embodiments, the expression of peptides (e.g., knotted peptides)as fusions with SCN can be utilized with an endogenously cleaving SCN,with RARYKR right after the CIDG, and an exogenously cleaved one, withENLYFQ in that position. The former can be cleaved by the mammaliancells during protein export (e.g., by furin), and the free SCN andknotted peptide can be secreted into surrounding media. ENLYFQ is atobacco etch virus (TEV) protease site, which is not found endogenouslyin mammalian cells. The constructs in this system can be secreted asfusions, allowing for the knotted peptide to be cleaved off later byadding exogenous TEV protease. This can be useful for recovering theknottins. In some embodiments, purification “handles” such aspoly-histidine or poly-arginine can be added to the SCN and subsequentlyremoved by proteolysis. In addition to the knotted peptides, thesefusion systems can also used for difficult-to-express proteins ofmedical interest such as chemokines, interleukins, and peptide hormones.

In some embodiments, the peptide comprises a knotted-peptide. In certainembodiments, the knotted-peptide is selected from the group consistingof chymotrypsin inhibitor, hefutoxin, bubble protein, the C-terminaldomain of midkine, and epiregulin. In some embodiments, the lipocalinprotein comprises siderocalin.

The lipocalin fusions (e.g., siderocalin and/or Lcn2 fused with aknotted peptide) can be used in several ways different ways. It could beused to increase the size of the target protein (for example a potentialtherapeutic) in order to increase its half-life. It could be used tosecrete the target protein where the target protein is naturallyexpressed in the cytoplasm. SCN also has unique ligand specificity andtightly binds catecholate siderophores (bacterial iron chelators). Thisopens the possibility of loading the SCN fusion with specific ligands,such as a chemotherapeutic or radioactive reagent or some type or acompound that has beneficial properties. SCN, when loaded withsiderophores and iron, has a deep red color that can aid inchromatography or other purification steps.

The lipocalin fusions of the present disclosure (e.g., SCN fused withHO-1) can be used in a variety of ways. For example, fusions can be usedto increase the size of the target protein (e.g., a potentialtherapeutic) in order to increase its biological half-life. Fusions canbe used to secrete the target protein where the target protein isnaturally expressed in the cytoplasm. Fusions can also be used to targetthe fusion partner protein to specific locations to maximize therapeuticeffects. SCN also has unique ligand specificity, tightly bindingcatecholate siderophores (bacterial iron chelators), and has kidneyprotective effects. Combining SCN with other kidney protective agents,like HO-1, which could generate synergistic functional effects. Thisalso opens the possibility of loading the SCN fusion with specificligands, such as radioactive metal atoms (e.g. ⁵⁵Fe or Th) to allow invivo tracking or specific cytotoxic activities. In addition to severalother advantages, the lipocalin fusion systems can be used to make largeamounts of protein over relatively short time frames. In someembodiments, the amount of peptide obtained can be less than about 10mg/L, less than about 20 mg/L, less than about 40 mg/L, less than about50 mg/L, less than about 100 mg/L, less than about 150 mg/L, less thanabout 180 mg/L, or less than about 200 mg/L. In some embodiments, theamount of peptide obtained can be between about 10 mg/L and 200 mg/L,between about 50 mg/L and 200 mg/L, between about 100 mg/L and 200 mg/L,and between about 150 mg/L and 200 mg/Lln some embodiments, the peptideis produced at a concentration less than about 500 mg/liter, less thanabout 400 mg/liter, less than about 300 mg/liter, less than about 200mg/liter, less than about 100 mg/liter, or less than about 50 mg/liter.

In other embodiments, some of the peptides described herein can beexpressed in a variety of ways known in the literature. For example, thepeptides are expressed in bacterial systems including E. Coli,corynebacterium, and pseudomonas fluoresceins. Expression platforms forE. coli can include periplasmic expression or cytoplasmic expression.For periplasmic expression, fusions can include pelB, dsbA, and ExFABPfusion. The peptides can also be expressed in insect cell systems andeukaryotic systems including mammalian systems.

In some aspects, the peptides disclosed herein can be introduced bytransfection, a technique that involves introduction of foreign DNA intothe nucleus of the eukaryotic cells. In some aspects, the peptides canbe synthesized by transient transfection (DNA does not integrate withthe genome of the eukaryotic cells, but the genes are expressed for24-96 hours). Various methods can be used to introduce the foreign DNAinto the host cells, and transfection can be achieved by chemical-basedmeans including by the calcium phosphate, by dendrimers, by liposomes,and by the use of cationic polymers. Non-chemical methods oftransfection include electroporation, sono-poration, opticaltransfection, protoplast fusion, impalefection, and hydrodynamicdelivery. In some embodiments, transfection can be achieved byparticle-based methods including gene gun where the DNA is coupled to ananoparticle of an inert solid which is then “shot” directly into thetarget cell's nucleus. Other particle-based transfection methods includemagnet assisted transfection and impalefection.

DNA can also be introduced into cells using virus as a carrier (viraltransduction) using reteroviruses or lentiviruses. In some embodiments,the peptides of the present invention can be prepared using a Daedalusexpression system. Ashok D. Bandaranayake et al., Nucleic Acids Res.2011 November; 39(21): e143, which is incorporated herein by referencein its entirety. This technique may also be combined with a serum freemammalian culture system. And, it is also possible to express taglessproteins, which can be purified in a single size exclusion step directlyfrom the media, at high levels.

In one aspect, the present invention provides a method of makinghundreds to thousands or more of peptide variants at high levels.Conventional methods of making knotted peptides can be limited in thatactivity of knotted peptides can depend on proper folding of thepeptides. There has been limited success in making knotted peptides thatfold properly during manufacture. The present invention overcomes theseproblems with other techniques known in the art. FIG. 2 shows an examplemethod for making the peptide libraries of the present invention. Asshown, viruses can be produced by packaging of specific oligonucleotidessequences, transferring the sequences to the viruses, and expressing thepeptides. Recovery and scale up of the peptides can be conducted, andthen the sample can be purified and assayed. The process can beconducted efficiently (e.g., in three weeks) and large amounts ofpeptide can be produced (e.g., 200 mg/liter). In some instances,purification by chromatography may not be needed due to the purity ofmanufacture according the methods described herein.

In an example embodiment, the present invention includes fusion proteinsof a knotted peptide fused to siderocalin via a cleavable linker. FIG. 3shows an example fusion system that can be used to make the knottedpeptide libraries. As shown, the fusion system includes a sequenceincluding an IgK SP, sFLAG, HIS, siderocalin, TEV, and the knottedpeptide sequence of interest. In some embodiments, these fusions can becombined with the Daedalus expression systems. Ashok D. Bandaranayake etal., Nucleic Acids Res. 2011 November; 39(21): e143, which isincorporated herein by reference in its entirety. A lentivirus can beused to gain rapid, stable expression in HEK293 cells, a human kidneycell line. The siderocalin can be highly expressed in this system and,e.g., serves to help the knotted peptide to be expressed as well. Thenature of the cleavable linker allows the fusion to be cleaved as theprotein is being expressed or later via an exogenously added protease.The siderocalin fusion partner can, e.g., be a generalizable expressionenhancement system for any difficult-to-express protein, can be used asa tag to increase the size of a smaller peptide, and/or to improve apeptide's serum half-life (e.g., by increasing the size of the finalfusion protein above the glomerular filtration limit. In someembodiments, the fusion protein further comprises at least one of an IgKstarter sequence, a sFLAG, a HIS, and a TEV. In certain embodiments, thefusion protein comprises the following construct: IgKSP-sFLAG-HIS-siderocalin-TEP-peptide. In some embodiments, the peptidecomprises a knotted-peptide.

Although HEK293 cells are robust and used for general proteinexpression, the lentivirus can infect a wide variety of cells. Combiningthis with a system that allows proteins to be cleaved as they areexpressed enables a set of powerful assays that rely upon the secretedpeptide to act in an autocrine or paracrine manner (i.e., they act onthe cell that is secreting them or on nearby cells). An example of thiswould be to infect cancer target cells with a library ofpeptide-expressing lentiviruses and then screen those cells by flowcytometry for those that showed signs of apoptosis (e.g., Annexin Vexpression). The cells showing signs of apoptotic stress could be sortedout and the viruses sequenced, essentially looking for cells that wereexpressing a peptide that was inducing apoptosis in an autocrinefashion. A related set of screens could be done in a diffusion-limitedmatrix (e.g., soft agar), where peptide-expressing cells were mixed withtarget cells and the agar limited diffusion of the peptide. Areas oftarget cell death would be an indication of an active secreted peptide.Screens done in this manner could employ very large libraries, as thedeconvolution would be as simple as sequencing the gene from which thepeptide came.

In some embodiments, the present invention can include methods forproducing knottins such that the knottin protein can remain tethered tothe surface of the mammalian cell for use in conventional bindingscreens (e.g., those in which the target molecule is tethered to acolumn or beads and candidate drugs are identified by affinity to thetarget). In contrast to other known methods (e.g., phage or yeastdisplay), the methods described herein use fusion systems (e.g., asiderocalin system of the present invention) to express libraries ofpeptides that have been designed according to the “rules” describedabove (e.g., ratio of acid/basic amino acids in a peptide) and that canbe established through the in vivo drug discovery process and/or thathave already been prescreened for specific biophysical andpharmacological properties. In these methods, e.g., all DNA sequencesand protein products are already known and have already been validated(e.g., the peptides all fold properly and have improved serum halflives). The methods of present invention are in direct contrast to otherknown display technologies where the displayed proteins are not knownand previously validated, and instead have their sequences randomized(using mutagenic oligonucleotides and degenerate NNN codons) yieldinglibraries of immense size (generally greater than 10⁷), where many ofthe proteins do not fold properly due to deleterious mutations.

Methods of Making Knotted Peptides and Related Compositions

The fusion systems of the present disclosure can be used in variousaspects for the production of peptides, knottins, and cytoplasmic andsecreted proteins. In some aspects, the methods and compositionsdescribed herein include fusion of target proteins and/or peptides tolipocalin such that lipocalin facilitates the expression and secretionof the target protein by a cell. For example, lipocalins have aconserved fold characterized by an eight-stranded beta barrel with aflanking alpha helix and supports a versatile scaffold. In some aspects,lipocalin fusion protein systems result in greater fusion proteinexpression in mammalian cells compared to systems without the use of alipocalin fusion protein. For example, lipocalin fusion protein systemsresult in less than 0.5 times greater, 1 times greater, 2 times greater,3 times greater, 4 times greater, 5 times greater, 6 times greater, 7times greater, 8 times greater, 9 times greater, 10 times greater, 11times greater, 12 times greater, 13 times greater, 14 times greater, 15times greater, 16 times greater, 17 times greater, 18 times greater, 19times greater, 20 times greater, 25 times greater, 30 times greater, 35times greater, 40 times greater, 45 times greater, 50 times greater, 55times greater, 60 times greater, 65 times greater, 70 times greater, 75times greater, 80 times greater, 85 times greater, 90 times greater, 95times greater, 100 times greater, 200 times greater, 300 times greater,400 times greater, 500 times greater, 600 times greater, 700 timesgreater, 800 times greater, 900 times greater, or 1000 times fusionprotein expression in mammalian cells compared to systems lacking thelipocalin fusion protein.

In some aspects, lipocalin (e.g., SCN) may be used as a fusion partnerto stabilize proteins or peptides of interest as immunogens. In aspects,the species from which the lipocalin sequence is derived is differentthan the recipient species. In some aspects, the species from which thelipocalin sequence is derived is the same as the recipient species.

SCN, and related proteins, when used as a secretion partneradvantageously improve the production of secreted proteins and peptides.Moreover, SCN, and related proteins, advantageously are small, therebyimproving their bioavailability. For example, the mature protein is 178amino acids and has a molecular weight of 20547 Da. SCN has a singleintramolecular disulfide bond, which increases its stability and asingle N-linked glycosylation site.

In some aspects, at least one of the amino acids in the native sequenceof lipocalin 2 (SCN) may be substituted for a non-native amino acid. Insome aspects, the mutations may be generated to prevent SCN fromdimerizing. For example, one SCN protein may dimerize with another SCNprotein at cysteine residues or one SCN protein may dimerize withdifferent proteins at cysteine residues. For example, generating a C87Smutation in SCN may prevent dimerization at a cysteine residue (seeGoetz, D. H., et al., ‘The Neutrophil Lipocalin NGAL is a BacteriostaticAgent that Interferes with Siderophore-mediated Iron Acquisition’Molecular Cell (2002) 10: 1033-43).

In some aspects, at least one of the amino acids in the native sequenceof the non-human lipocalin protein orthologous to the human SCN may besubstituted for a non-native amino acid. For example, non-humanorthologs of the human lipocalin protein that may be used with themethods and compositions described herein include, but are not limitedto, murine Lcn2 (e.g., 24p3), Lcn1, Lcn6, Lcn8, Lcn9, Lcn10, Lcn12 andLcn15 (see FIG. 14). In some aspects, the mutations may be generated toprevent non-human lipocalin protein orthologous to the human SCN fromdimerizing. For example, one non-human lipocalin protein orthologous tothe human SCN protein may dimerize with another non-human lipocalinprotein orthologous to the human SCN protein at cysteine residues or onenon-human lipocalin protein orthologous to the human SCN protein maydimerize with a different protein at cysteine residues. For example,generating a mutation at a site orthologous to the C87S mutation inhuman lipocalin protein may prevent dimeraizaiton at a cysteine residue(see Goetz, D. H., et al., ‘The Neutrophil Lipocalin NGAL is aBacteriostatic Agent that Interferes with Siderophore-mediated IronAcquisition’ Molecular Cell (2002) 10: 1033-43).

In an exemplary aspect, a lipocalin fusion protein may contain thefollowing protein sequence:

MPLGLLWLGLALLGALHAQAQDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGCQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDGIn this sequence, the annotations indicate, a signal peptide,glycosylation site, disulfide bond and the C87S mutation.

In some aspects, fusion of a protein or peptide of interest to lipocalinmay improve the biological properties of the target protein (e.g., apotential therapeutic). For example, fusion of a protein or peptide ofinterest to lipocalin may increase the half-life of the protein orpeptide of interest by increasing the size of the overall protein. Forexample, the increased size of the protein may prevent glomerularfiltration of the protein or peptide of interest. In some aspects,fusions of small proteins, such as antibody fragments, exhibit decreasedglomerular filtration. These effects are observed with both enzymes andFab antibody fragments. In some embodiments, the fusion protein furthercomprises a peptide selected from the group consisting of: an IgKstarter sequence, a sFLAG, a HIS, siderocalin, a TEV and the knottedpeptide sequence of interest. In certain embodiments, the fusion proteincomprises the following construct: IgKSP-sFLAG-HIS-siderocalin-TEV-peptide. In some embodiments, theknotted-peptide includes at least two disulfide bonds.

In some embodiments, the fusion protein further comprises at least oneof an IgK starter sequence, a sFLAG, a HIS, and a TEV. In certainembodiments, the fusion protein comprises the following construct: IgKSP-sFLAG-HIS-siderocalin-TEV-peptide. In some embodiments, the fusionprotein is generated by direct fusion of each subunit to the adjacentsubunits. In certain embodiments, the composition further comprises alinker sequence between the peptide or protein domain and the lipocalinprotein. In some embodiments, the peptide comprises a knotted-peptide.

In some embodiments, the fusion protein further comprises at least oneof an IgK starter sequence, a sFLAG, a HIS, and a TEV. In certainembodiments, the fusion protein comprises the following construct: IgKSP-sFLAG-HIS-siderocalin-TEV-peptide. In other embodiments, the peptidecomprises a knotted-peptide

In some aspects, fusion of the protein or peptide of interest tolipocalin may enhance the purification of the protein or peptide ofinterest after production. For example, the protein or peptide ofinterest fused to lipocalin may be produced from a protein expressionsystem (e.g., fusion protein expression system). For example, proteinsor peptides of interest may be retained in a compartment of a cellduring production if proteins or peptides of interest are not fused tolipocalin.

In some aspects, SCN has protective properties in vivo that are impartedto a fusion partner as the basis of in vivo therapeutics fusionpartners. In some aspects, these therapeutics can be stabilized for useas immunogens, matching the species donating the lipocalin sequence tothat of the recipient species to focus elicited immune responses to thefusion partner. SCN also has unique ligand specificity and tightly bindssiderophores (ferric iron chelators).

The methods and compositions described herein include fusion proteins ofa protein or peptide of interest coupled to SCN and a SCN ligand. Insome aspects, the ligands for SCN may include, but are not limited to, asiderophore or an organizing metal. In some aspects, fusion proteinsincluding the SCN ligands may be coupled to a fluorphore. For example,the coupling may be covalently coupled. In some aspects, fusion proteinsincluding the SCN ligands may be coupled to a luminescent siderophore.In some aspects, the luminescent siderophore may include a metalcomplex.

In some aspects, the addition of SCN ligands to the fusion proteinscould be used to detect or localize the target protein, or targetpeptide, of the fusion protein. For example, the detection andlocalization could be performed either in vitro or in vivo. In someaspects, the addition of SCN ligands to the fusion proteins could beused to purify fusion proteins from mixtures. For example, an SCN ligand(e.g., a siderophore/metal complex) could be contacted with at least onepurification resin metal other than, including but not limited to,aluminum, gadolinium, indium, vanadium, plutonium or thorium, and anyrelated isotopes.

In some aspects, the addition of SCN ligands to the fusion proteinscould be used for the delivery of radionuclides to a target tissue. Insome aspects, the addition of SCN ligands to the fusion proteins couldbe used to deliver iron to a target. In addition, SCN bound tosiderophores and iron can be a dark red color. For example, the dark redcolor can be combined with methods and compositions of chromatography orother steps in a method of purification.

In an exemplary aspect, a self-cleaving SCN isoform may be added to apeptide or protein of interest to generate a fusion protein. Forexample, the self-cleaving SCN isoform may contain the RARYKR amino acidsequence immediately following the CIDG amino acid sequence. In thiscase, the RARYKR sequence may be cleaved by an endogenous enzyme to thecells of the protein expression system (e.g., the mammalian cells)during export of the fusion protein. For example, furin may cleave theRARYKR sequence. In this case, SCN and the peptide or protein ofinterest may be free and located in the extracellular space.

In an exemplary aspect, an exogenously cleaved SCN isoform may be addedto a peptide or protein of interest to generate a fusion protein. Forexample, the exogenously cleaved SCN isoform may contain the ENLYFQamino acid sequence immediately following the CIDG amino acid sequence.In this case, the ENLYFQ sequence may be cleaved by an exogenous enzymeto the cells of the protein expression system (e.g., the mammaliancells) during export or after export of the fusion protein. For example,a tobacco etch protease may cleave the ENLYFQ site.

In some aspects, the fusion proteins may be secreted from the cells ofthe protein expression system (e.g., the mammalian cells) as fusionproteins. In this case, the peptide or protein of interest may becleaved from the SCN protein by TEV protease. For example, the TEVprotease may be added to the cells of the protein expression system(e.g., the mammalian cells) or added after removal of the fusion proteinfrom the cells.

In some aspects, the SCN peptide may be modified to include compoundsfor purification or isolation. In some aspects, the compounds may be anamino acid or more than one amino acid. For example, the compounds maybe poly-histidine or poly-arginine and may be located between Lcn2 and asignal peptide in the fusion protein. In some aspects, the compounds maybe removed from Lcn2 using an enzyme or proteolysis.

The present invention relates to compositions comprising a peptidelibrary, the peptide library further comprising a plurality of peptideslacking at least one native lysine residue. In some embodiments, thepeptides are conjugated to an adaptor molecule. In certain embodiments,the adaptor molecule is a peptide. In some embodiments, the peptide hasa unique signature determined by mass spectroscopy.

Methods of Use

The fusion proteins, peptides, or conjugates thereof of the presentdisclosure can be used for a variety of other applications, such astherapeutic and/or diagnostic applications. In some embodiments, thefusion proteins, peptides, or conjugates thereof of the presentdisclosure can be used for methods of treating diseases. In someembodiments, the fusion proteins, peptides, or conjugates thereof of thepresent disclosure can be used to deliver drugs to, e.g., tumors in thebrain of a subject.

The present invention also provides compositions for administering thefusion proteins, peptides, or conjugates thereof described herein to asubject to facilitate diagnostic and/or therapeutic applications.

In certain embodiments, the compositions can include a pharmaceuticallyacceptable excipient. Pharmaceutical excipients useful in the presentinvention include, but are not limited to, binders, fillers,disintegrants, lubricants, coatings, sweeteners, flavors and colors. Oneof skill in the art will recognize that other pharmaceutical excipientsare useful in the present invention. The term “pharmaceuticalcomposition” as used herein includes, e.g., solid and/or liquid dosageforms such as tablet, capsule, pill and the like.

The fusion proteins, peptides, or conjugates thereof of the presentdisclosure may be administered by any suitable technique available inthe art, e.g., as compositions. For example, they can be administered asfrequently as necessary, including hourly, daily, weekly or monthly. Thefusion proteins, peptides, or conjugates thereof, can be utilized in themethods of the invention can be, e.g., administered at dosages that maybe varied depending upon the requirements of the method being employed.The fusion proteins, peptides, or conjugates thereof described hereincan be administered to the subject in a variety of ways, includingparenterally, subcutaneously, intravenously, intratracheally,intranasally, intradermally, intramuscularly, colonically, rectally,urethrally or intraperitoneally. In some embodiments, the pharmaceuticalcompositions can be administered parenterally, intravenously,intramuscularly or orally. In some embodiments, the fusion proteins,peptides, or conjugates thereof of the present disclosure, can beadministered systemically. In some embodiments, the compositions can beadministered intratumorally and/or intranodally, such as delivery to asubject's lymph node(s). In certain embodiments, administration caninclude enteral administration including oral administration, rectaladministration, and administration by gastric feeding tube or duodenalfeeding tube. Administration can also be including intravenousinjection, intra-arterial injection, intra-muscular injection,intracerebral, intracerebroventricular or subcutaneous (under the skin)administration. In some embodiments, administration can be achieved bytopical means including epicutaneous (application to skin) andinhalation.

The oral agents comprising fusion proteins, peptides, or conjugatesthereof of the present disclosure can be in any suitable form for oraladministration, such as liquid, tablets, capsules, or the like. The oralformulations can be further coated or treated to prevent or reducedissolution in stomach. The compositions of the present invention can beadministered to a subject using any suitable methods known in the art.Suitable formulations for use in the present invention and methods ofdelivery are generally well known in the art. For example, the fusionproteins, peptides, or conjugates thereof of the present disclosure canbe formulated as pharmaceutical compositions with a pharmaceuticallyacceptable diluent, carrier or excipient. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions including pH adjusting andbuffering agents, tonicity adjusting agents, wetting agents and thelike, such as, for example, sodium acetate, sodium lactate, sodiumchloride, potassium chloride, calcium chloride, sorbitan monolaurate,triethanolamine oleate, etc.

As used herein, a “subject” is a human or non-human animal. In someembodiments, a subject can include, but is not limited to, a mouse, arat, a rabbit, a human, or other animal. In another embodiment, asubject is a human, such as a human having or at risk of having acancer. In some embodiments, a subject or biological source may besuspected of having or being at risk for having a disease, disorder orcondition, including a malignant disease, disorder or condition (e.g.,cancer). In certain embodiments, a subject or biological source may besuspected of having or being at risk for having a hyperproliferativedisease (e.g., carcinoma, sarcoma), and in certain other embodiments ofthis disclosure a subject or biological source may be known to be freeof a risk or presence of such disease, disorder, or condition.

“Treatment,” “treating” or “ameliorating” refers to either a therapeutictreatment or prophylactic/preventative treatment. A treatment istherapeutic if at least one symptom of disease (e.g., ahyperproliferative disorder, such as cancer) in an individual receivingtreatment improves or a treatment may delay worsening of a progressivedisease in an individual, or prevent onset of additional associateddiseases (e.g., metastases from cancer).

A “therapeutically effective amount (or dose)” or “effective amount (ordose)” of a composition including fusion proteins, peptides, orconjugates thereof of the present disclosure, refers to that amount ofcompound sufficient to result in amelioration of one or more symptoms ofthe disease being in a statistically significant manner. When referringto an individual active ingredient, administered alone, atherapeutically effective dose refers to that ingredient alone (e.g., afusion proteins, peptides, or conjugates thereof of the presentdisclosure). When referring to a combination, a therapeuticallyeffective dose refers to combined amounts of the active ingredients thatresult in the therapeutic effect, whether administered serially orsimultaneously (in the same formulation or in separate formulations).

The term “pharmaceutically acceptable” refers to molecular entities andcompositions that do not produce allergic or other serious adversereactions when administered to a subject using routes well known in theart.

A “patient in need” or “subject in need” refers to a patient or subjectat risk of, or suffering from, a disease, disorder or condition (e.g.,cancer) that is amenable to treatment or amelioration with a fusionproteins, peptides, or conjugates thereof of the present disclosuredescribed herein.

In some embodiments, the fusion proteins, peptides, or conjugatesthereof of the present disclosure can further include other agents tofacilitate treatment. For example, a fusion proteins, peptides, orconjugates thereof of the present disclosure can further includecytotoxic agents (e.g., mitotic inhibitors), toxins, antisensenucleotides, cancer treatment drugs (e.g., alkylating agents),nucleotide drugs, anti-metabolites, metabolic modulators,radiosensitizers, peptide therapeutics, peptide-drug conjugates,radionuclides, or a combination thereof.

Cytotoxic agents can include drugs that can be used to treat cancer,e.g., by inhibiting cell proliferation. Some example cytotoxic agentscan include, e.g., the vinca alkaloids, mitomycins, bleomycins,cytotoxic nucleosides, taxanes, and epothilones, Members of thoseclasses include, for example, doxorubicin, carminomycin, daunorubicin,aminopterin, methotrexate, methopterin, dichloromethotrexate, mitomycinC, porfiromycin, 5-fluorouracil, 6-mercaptopurine, gemcitabine, cytosinearabinoside, podophyllotoxin or podo-phyllotoxin derivatives, such asetoposide, etoposide phosphate or teniposide, melphalan, vinblastine,vincristine, leurosidine, vindesine, leurosine, paclitaxel andtherapeutically effective analogs and derivatives of the same. Otheruseful antineoplastic agents include estramustine, cisplatin,carboplatin, cyclophosphamide, bleomycin, gemcitibine, ifosamide,melphalan, hexamethyl melamine, thiotepa, cytarabin, idatrexate,trimetrexate, dacarbazine, L-asparaginase, camptothecin, CPT-11,topotecan, ara-C, bicalutamide, flutamide, leuprolide, pyridobenzoindolederivatives, interferons and interleukins.

Suitable metabolic modulators can include, but are not limited to,lonidamine, dichloroacetate, alpha-tocopheryl succinate, methyljasmonate, betulinic acid, and resveratrol

Radiosensitizers are known to increase the sensitivity of cancerouscells to the toxic effects of electromagnetic radiation, e.g., x-rays.Examples of x-ray activated radiosensitizers include, but are notlimited to, metronidazole, misonidazole, desmethylmisonidazole,pimonidazole, etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233,E09, RB 6145, nicotinamide, 5-bromodeoxyuridine (BUdR),5-iododeoxyuridine (I UdR), bromodeoxycytidine, fluorodeoxyuridine(FudR), hydroxyurea, cisplatin, and therapeutically effective analogsand derivatives of the same.

In some embodiments, the fusion proteins, peptides, or conjugatesthereof of the present disclosure can include radionuclides and/orcomplexed radionuclides. Suitable radionuclides can include, but are notlimited to, Sc-47, Ga-67, Y-90, Ag-111, In-111, Sm-153, Tb-166, Lu-177,Bi-213, Ac-225, Cu-64, Cu-67, Pd-109, Ag-111, Re-186, Re-188, Pt-197,Bi-212, Bi-213, Pb-212 or Ra-223.

In certain embodiments, the present invention can include treatingdiseases, disorders, and/or conditions, such as gliomas, astrocytomasmedulloblastomas, choroids plexus carcinomas, ependymomas, other braintumors, neuroblastoma, head and neck cancer, lung cancer, breast cancer,intestinal cancer, pancreatic cancer, liver cancer, kidney cancer,sarcomas, osteosarcoma, rhabdomyosarcoma, Ewing's sarcoma, carcinomas,melanomas, ovarian cancer, cervical cancer, lymphoma, thyroid cancer,anal cancer, colo-rectal cancer, endometrial cancer, germ cell tumors,laryngeal cancer, multiple myeloma, prostate cancer, retinoblastoma,gastric cancer, testicular cancer, and Wilm's tumor. In someembodiments, the methods can including treating a disease, disorderand/or condition including a glioma, a skin cancer, a lung cancer, alymphoma, a medulloblastoma, a prostate cancer, a pancreatic cancer, ora combination thereof. In certain embodiments, the methods can be usedto treat breast and mammary cancers, colon, skin, lung, lymphoma,glioma, medulloblastoma prostate, pancreatic cancers, oral squamous cellcarcinoma, and/or hemangiopericytoma.

The present invention further includes methods of administering a fusionproteins, peptides, or conjugates thereof of the present disclosure. Forexample, in one aspect, the present invention includes a methodcomprising a step of administering an effective dose of a fusionproteins, peptides, or conjugates thereof of the present disclosure or acomposition including fusion proteins, peptides, or conjugates thereofof the present disclosure to a subject with a tumor such that thepeptide selectively targets tumor tissue over normal tissue.

The methods can further include facilitating surgical removal ofcancerous tissue (e.g., a tumor) in a subject. For example, the presentinvention can include a method comprising administering an effectivedose of fusion proteins, peptides, or conjugates thereof of the presentdisclosure or a composition including fusion proteins, peptides, orconjugates thereof of the present disclosure to a subject with canceroustissue (e.g., a tumor) such that the peptide selectively targetscancerous tissue (e.g., tumor tissue) over normal tissue. The methodscan include imaging the cancerous tissue by, e.g., detecting the tissuethat shows elevated binding of the peptides, thereby indicating thelocation of the cancerous tissue. Identification of the location canprovide a step of surgically removing the cancerous tissue from thesubject. The surgically removing can include, e.g., intraoperativevisualization of the cancerous tissue as identified by binding of thefusion proteins, peptides, or conjugates thereof of the presentdisclosure.

The present invention also provides compositions for administeringfusion proteins, peptides, or conjugates thereof of the presentdisclosure to a subject to facilitate diagnostic and/or therapeuticapplications. In certain embodiments, the compositions can include apharmaceutically acceptable excipient. Pharmaceutical excipients usefulin the present invention include, but are not limited to, binders,fillers, disintegrants, lubricants, coatings, sweeteners, flavors andcolors. One of skill in the art will recognize that other pharmaceuticalexcipients are useful in the present invention. The term “pharmaceuticalcomposition” as used herein includes, e.g., solid and/or liquid dosageforms such as tablet, capsule, pill and the like.

The fusion proteins, peptides, or conjugates thereof of the presentdisclosure can be administered as frequently as necessary, includinghourly, daily, weekly or monthly. The fusion proteins, peptides, orconjugates thereof of the present disclosure utilized in the methods ofthe invention can be, e.g., administered at dosages that may be varieddepending upon the requirements of the method being employed. The fusionproteins, peptides, or conjugates thereof of the present disclosure canbe administered to the subject in a variety of ways, includingparenterally, subcutaneously, intravenously, intratracheally,intranasally, intradermally, intramuscularly, colonically, rectally,urethrally or intraperitoneally. In some embodiments, the pharmaceuticalcompositions can be administered parenterally, intravenously,intramuscularly or orally. In some embodiments, the compositions can beadministered intratumorally and/or intranodally, such as delivery to asubject's lymph node(s). In certain embodiments, administration caninclude enteral administration including oral administration, rectaladministration, and administration by gastric feeding tube or duodenalfeeding tube. Administration can also be including intravenousinjection, intra-arterial injection, intra-muscular injection,intracerebral, intracerebroventricular or subcutaneous (under the skin)administration.

The oral agents comprising peptides or protein fusions described hereincan be in any suitable form for oral administration, such as liquid,tablets, capsules, or the like. The oral formulations can be furthercoated or treated to prevent or reduce dissolution in stomach. Thecompositions of the present invention can be administered to a subjectusing any suitable methods known in the art. Suitable formulations foruse in the present invention and methods of delivery are generally wellknown in the art. For example, the peptides or fusion proteins describedherein can be formulated as pharmaceutical compositions with apharmaceutically acceptable diluent, carrier or excipient. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions includingpH adjusting and buffering agents, tonicity adjusting agents, wettingagents and the like, such as, for example, sodium acetate, sodiumlactate, sodium chloride, potassium chloride, calcium chloride, sorbitanmonolaurate, triethanolamine oleate, etc.

The present invention further includes functional assays of fusionproteins, peptides, or conjugates thereof of the present disclosure. Thecapacity of fusion proteins, peptides, or conjugates thereof of thepresent disclosure, to bind to tumor or cancerous tissue can be assayedby in vitro binding, ex vivo imaging, animal models, and other assaysknown in the art and as previously described. See, for example, USPatent Publication Number US20080279780 and WO 2011/142858, both ofwhich are incorporated by reference herein for the description offunctional assays to detect and measure binding to tumor cells and tumortissue.

One skilled in the art will be knowledgeable about animal models thatare useful for measuring the in vivo activity of fusion proteins,peptides, or conjugates thereof of the present disclosure. For example,the National Cancer Institute maintains a database of specific cancermodels. See the “Cancer Models Database” at the National CancerInstitute website. All animals are handled in strict accordance with theNational Institutes of Health Guide for the Care and Use of LaboratoryAnimals. ND2:SmoA1 medulloblastoma mice, TRAMP prostate cancer mice andApc^(1638N) intestinal adenoma and adenocarcinoma mice have beenpreviously described. See, Fodde, R., et al., A targetedchain-termination mutation in the mouse Apc gene results in multipleintestinal tumors. Proc. Natl. Acad. Sci. U.S.A., 1994. 91(19): p.8969-73; Greenberg, N. M., et al., Prostate cancer in a transgenicmouse. Proc. Natl. Acad. Sci. U.S.A., 1995. 92(8): p. 3439-43;Kaplan-Lefko, P. J., et al., Pathobiology of autochthonous prostatecancer in a pre-clinical transgenic mouse model. Prostate, 2003. 55(3):p. 219-37; Hallahan, A. R., et al., The SmoA1 mouse model reveals thatnotch signaling is critical for the growth and survival of sonichedgehog-induced medulloblastomas. Cancer Res., 2004. 64(21): p.7794-800; each expressly incorporated herein by reference in itsentirety.

The fusion proteins, peptides, and conjugates therof generated andproduced by the methods and systems described herein can be used for arange of applications. For example, the proteins and peptides can beused for therapeutic and/or diagnostic purposes. Some example usesinclude, but are not limited to, conjugating the fusion proteins orpeptides to radiolabels and/or fluorescent molecules for bioimaging,linking the peptides to cytotoxic agents, using the peptides for invitro diagnostics for biochemical assays, as well as, e.g., forveterinary uses, insecticides, antibiotics, herbicides, antifreezecompositions, and antivenoms.

As will be appreciated by one of ordinary skill in the art, the fusionproteins and peptides described herein can be tailored for a wide rangeof targets (e.g., therapeutic targets). In some embodiments, the targetsare associated with a variety of diseases or disorders. Some targets,for example, can include but are not limited to glypican-2 (GPC2),protocadherin (1□(PCDHA1), Ca_(v)2.2, K_(v)1.3, Na_(v)1.2, NaV1.1,NaV1.7, NaV1.8, CIC-3, nAChR, NMDA-R, NPRA, GLP-1R, □_(1B)-AR, NT-R-1,ACE, NET mTor, cMet, VEGF/VEGFR, c-Kit, PDGF/PDGFR, PI3K, HER2, EGFR,Orai1, CD47, Raf, NF□B, Bromodomains, HATS, HDAC, LDH, IDH2, CD22, MIC,c-Myc, n-Myc, PHFSA, BUB1B, Bcl-2, k-Ras, Notch1, p53, α5β3, NKG2D,CTLA4/CD28, and/or Mcl-1.

The present invention also provides compositions for administering thefusion peptides, peptides, or conjugates thereof according to thepresent disclosure to a subject to facilitate diagnostic and/ortherapeutic applications. In certain embodiments, the compositions caninclude a pharmaceutically acceptable excipient. Pharmaceuticalexcipients useful in the present invention include, but are not limitedto, binders, fillers, disintegrants, lubricants, coatings, sweeteners,flavors and colors. One of skill in the art will recognize that otherpharmaceutical excipients are useful in the present invention. The term“pharmaceutical composition” as used herein includes, e.g., solid and/orliquid dosage forms such as tablet, capsule, pill and the like.

The fusion peptides, peptides, or conjugates thereof according to thepresent disclosure can be administered as frequently as necessary,including hourly, daily, weekly or monthly. The fusion peptides,peptides, or conjugates thereof according to the present disclosureutilized in the methods of the invention can be, e.g., administered atdosages that may be varied depending upon the requirements of the methodbeing employed. The fusion peptides, peptides, or conjugates thereofaccording to the present disclosure can be administered to the subjectin a variety of ways, including parenterally, subcutaneously,intravenously, intratracheally, intranasally, intradermally,intramuscularly, colonically, rectally, urethrally or intraperitoneally.In some embodiments, the pharmaceutical compositions can be administeredparenterally, intravenously, intramuscularly or orally. In someembodiments, the fusion peptides, peptides, or conjugates thereofaccording to the present disclosure can be administered systemically. Insome embodiments, the compositions can be administered intratumorallyand/or intranodally, such as delivery to a subject's lymph node(s). Incertain embodiments, administration can include enteral administrationincluding oral administration, rectal administration, and administrationby gastric feeding tube or duodenal feeding tube. Administration canalso be including intravenous injection, intra-arterial injection,intra-muscular injection, intracerebral, intracerebroventricular orsubcutaneous (under the skin) administration. In some embodiments,administration can be achieved by topical means including epicutaneous(application to skin) and inhalation.

The oral agents comprising a fusion peptides, peptides, or conjugatesthereof according to the present disclosure described herein can be inany suitable form for oral administration, such as liquid, tablets,capsules, or the like. The oral formulations can be further coated ortreated to prevent or reduce dissolution in stomach. The compositions ofthe present invention can be administered to a subject using anysuitable methods known in the art. Suitable formulations for use in thepresent invention and methods of delivery are generally well known inthe art. For example, the fusion peptides, peptides, or conjugatesthereof according to the present disclosure can be formulated aspharmaceutical compositions with a pharmaceutically acceptable diluent,carrier or excipient. The compositions may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions including pH adjusting and buffering agents, tonicityadjusting agents, wetting agents and the like, such as, for example,sodium acetate, sodium lactate, sodium chloride, potassium chloride,calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

EXEMPLARY ASPECTS Example 1 Expressing Peptide Constructs for KnottinGeneration

This example describes a method for expressing peptide constructs inculture and greatly facilitating their development, particularly asdrugs.

As shown in FIG. 5, the various knottins can be expressed, e.g., in alentivirus expression-based method that can include packaging, transfer,and then expression followed by isolation and/or purification of theexpressed knottin peptides. Several coding constructs can be used. Inthis example, the encoding of the knottin peptides included apolynucleotide construct including IgKSP-sFLAG-HIS-Siderocalin-TEV-Knottin. Specific sequences of some exampleconstructs are disclosed in the “SEQUENCE” section below. FIG. 6 showsgel data of a number of example knottins that were made according to themethod described in FIG. 5. As shown, chymotrypsin inhibitor (CTI),epiregulin (EPI), hefutoxin (HTX), bubble protein (BUB), potatocarboxypeptidase inhibitor (PCI) were properly folded.

FIG. 9 shows a schematic describing production of a pooled library ofknottins. In this example, sequences of thousands of knottins can beencoded in an oligonucleotide pool (1) and selectively amplified usingunique primer pairs (2). DNA sublibraries can be cloned into theexpression vector, which results in the knottin variants that can, e.g.,have unique parental mass signatures and unique tryptic fragment masssignatures that can be resolvable using current techniques, such as massspectroscopy.

FIG. 8 includes example knottin variants that describe representativesequencing from a cloned knottin library. The sequences show rawsequencing data from a single round of library cloning. The sequenceportions highlighted in grey are full length knottin variants, and theerrors in oligonucleotide synthesis can explain the truncated andextended peptide sequences.

Using the methods described in this example, variants of several knottinscaffolds were generated and analyzed. FIG. 9 shows an SDS-PAGE analysisof 3000 member knottin libraries for, e.g., hefutoxin, knottin andchymotrypsin inhibitor. Each column of the SDS-PAGE gel shows a purifiedsample of a pool of 3000 knottin protein variants run under native andreducing conditions. The migration shift between the paired bandsindicates disulfide formation.

Four scaffolds were selected for the generation of defined libraries:hefutoxin, CTI, knottin, and epiregulin. A list of target amino acidsequences was generated in silico such that every member of each librarywould have a tryptic fragment with a unique mass; mutations wereselected to be structurally adjacent in order to generate bindingepitopes. The cysteines were not mutated, and lysine was specificallyavoided in order to make N-terminal conjugation unambiguous. 3000variants of each scaffold were generated, and each scaffold was flankedby a unique set of PCR primer sites so that each of the foursublibraries could be amplified independently. All constructs had anN-terminal BamHI site and a C-terminal NotI site, and following PCRamplification of each sublibrary from the pool of 12000oligonucleotides, each sublibrary was restriction digested and clonedinto cut parental vector (both the furin-cleaved and TEV-cleavedversions) as an SCN fusion protein using standard techniques. HEK293cells were transfected with this plasmid library as well as theaccessory plasmids needed for Daedalus expression, and the virus in themedia harvested 3-4 days later. Virus was concentrated by centrifugationand used to infect HEK293 cells for protein production using standardprocedures. We have found that the TEV-cleavable construct istechnically easier to handle when producing libraries because it allowsfor facile recovery of the fusion by IMAC on nickel resin. FollowingIMAC, the fusion protein was dialyzed into PBS and allowed to cleaveovernight with 6×His tagged TEV protease, and the SCN and protease weresubsequently removed by running the material through nickel resin again.The flow-through containing the cleaved peptide libraries was furtherpurified and buffer exchanged by size exclusion chromatography (SEC)into 10 mM ammonium formate, and the fractions containing the peptideswere pooled and lyophilized.

There were two approaches taken to cloning, Seamless Cloning(Invitrogen) and restriction/ligation based methods. Seamless cloningwas employed for making single constructs, typically using synthesized“gBlocks” from IDT. The manufacturer's instructions were followed.Restriction/ligation methods were standard and were used for cloninglibraries as follows: the pooled oligonucleotides from CustomArray weresubjected to PCR in order to amplify the relevant sublibrary. Theamplified pool was agarose gel purified and cleaned of agarose using aQiagen column. The purified fragment was digested with FaastDigest(Fermentas) BamHI and NotI and ligated into the parental vector whichhad been cut with the same two restriction endonucleases. Singletonclones were sequence verified, and 48 members of each library weresequenced in order to verify library quality.

The cloned knottin or library was cotransfected into HEK293 cells andmedia was collected as described (“Daedalus: A Robust, Turnkey Platformfor Rapid Production of Decigram Quantities of Active RecombinantProteins in Human Cell Lines Using Novel Lentiviral Vectors.”Bandaranayake A. D., et al., Nucleic Acids Res. (2011) 39(21):e143).Fusion protein was isolated using nickel IMAC and cleaved withrecombinant TEV protease. Excess siderocalin was removed via sizeexclusion chromatography, a process which also allowed the buffer to beswitched to 10 mM ammonium formate. The knottin containing fractionswere then lyophilized. Proper folding and peptide uniformity wasdemonstrated via SEC chromatography, reverse-phase HPLC, massspectrometry, and a gel shift in reduced versus non-reduced samples inSDS-PAGE.

Conjugation to palmitic acid, ICG, or biotinidase-resistant biotin wasperformed using a 3-10 fold excess of commercially available, activatedester conjugate in PBS. Acetonitrile was added when there weresolubility problems. The final material was purified by RP-HPLC forsingletons, and excess conjugate was removed from libraries by dialysis.

Example 2 Fusion Protein Systems

This example describes expression systems for the efficient productionof various peptides, including knotted peptides. Advantageously, thepeptides produced according to these methods are secreted and stable.

Siderocalin can be used according to the present disclosure forconstruction of a Scn-peptide fusion, which can be secreted and cleavedfor the efficient production of excreted peptides. FIG. 24 depicts thecrystal structure of Scn with a Th ligand.

FIG. 14 shows the alignment of Scn sequences from 18 species. Thealignment shows a high level of sequence conservation between the 18species with recognizable orthologs. Positions with an asterisk arepossible cites for ligand binding.

FIG. 15 depicts a 3D model of super-stable Scn in accordance with anaspect of the present disclosure. In this form of Scn, a seconddisulfide bond was engineered in order to secure the N-terminus andincrease thermal stability.

FIGS. 10-12 depict various manufacturing methods according to thepresent disclosure, which enable the efficient production of knottinpeptides. FIG. 10 depicts the elements of the siderocalin fusion proteinand use thereof for the production of knottin peptides. In this example,the knottin is secreted as a fusion protein to the siderocalin. The IgKsignal peptide is derived from the mouse Light Chain IgG. The sFLAG isthe short FLAG (DYKDE) for enhanced cleavage of the signal peptiderelative to the long FLAG signal peptide. The His is a histidine tag(HHHHHH) and TEV is the tobacco etch virus protease site (ENLYFQ).According to this method, and as depicted in FIG. 12, the fusion proteinis secreted, followed by cleavage and isolation. IgK SP is Murine IgKlight chain signal peptide, sFLAG: Shortened FLAG epitope, HIS: 6×histidine tag, TEV: Tobacco Etch Virus Protease recognition site andFurin—furin cleavage site with BamHI site.

FIG. 10 depicts the elements of the siderocalin fusion protein and theuse thereof for the production of knottin peptides. In this example, theknottin is secreted as a fusion protein to HIS-tagged siderocalin. TheHIS tag is six histidines (HHHHHH) that will reversibly bind nickel andis therefore a useful purification tag for Immobilized MetalChromatography (IMAC). Following isolation of the fusion protein byIMAC, the fusion is cleaved with TEV protease and the cleavedsiderocalin removed by another round of IMAC, leaving pure knottin.

FIG. 11 depicts the elements of the siderocalin fusion protein and usethereof for the production of knottin peptides. This method includes thesecretion of a siderocalin fusion with concomitant, intracellularcleavage at the Furin-His junction and purification by IMAC. The HIS tagcan then be removed from the knottin with TEV protease. The secretionmethod depicted in FIG. 11 utilizes the same restriction sites as themethod depicted in FIG. 10.

FIG. 12 depicts the elements of a system for the secretion of cleavedknottin peptides. The siderocalin in this case is present duringtranslation but is cleaved off by intracellular furin during protein.The secretion method depicted in FIG. 12 utilizes the same restrictionsites as the method depicted in FIG. 10.

FIG. 16 shows a schematic of a generic Scn fusion that can be used invarious aspects of the present disclosure. The native signal peptide wasremoved and an exogenous sFLAG and HIS tag were added to facilitatepurification. These modifications are optionally present in variousaspects of the disclosure.

FIG. 13 depicts exemplary knottins made using the Daedalus system andthe corresponding SDS PAGE analyses in accordance with an aspect of thepresent disclosure. The Dadalus system is desribed in Bandaranayake A.D., et al., Nucleic Acids Res. (2011) 39(21):e143. SDS PAGE analyseswere performed under reducing (left) and non-reducing (right)conditions. As shown in FIG. 13, a number of challenging peptides can beproduced according to the presently described methods.

FIG. 17 depicts a schematic of a light chain antibody fusion with Scnand a corresponding SDS PAGE analysis according to an aspect of thepresent disclosure. The Scn fusion enables the generation of Fabfragments that have an increased molecular weight (˜75 kDa) and improvedserum half-life. The addition of the Scn moiety also enables thedelivery of an exogenous ligand through the Scn ligand binding site.Also depicted is the SDS PAGE analysis under non-reducing and reducingconditions of a construct according to one aspect of the presentdisclosure.

FIG. 18 shows the SDS PAGE analysis comparing the expression of a Scnfusion with a cytoplasmic enzyme, HMOX1, both before and after cleavageaccording to an aspect of the present dislcosure. By cleaving the fusionprotein as shown in FIG. 18, it is demonstrated that the HMOX1 proteinis stable even after separation from Scn. This result further suggeststhat, using the presently described methods, it is possible to expressenzymes in a mammalian cell culture system where they are secreted outof the cell.

FIG. 19 depicts the expression of a Scn fusion with a cytoplasmic viralprotein Adv2 and the corresponding SDS PAGE analysis according to oneaspect of the present disclosure. By cleaving the fusion protein asshown in FIG. 19, it is demonstrated that the Adv2 protein is stableeven after separation from Scn. This result further suggests that, usingthe presently described methods, it is possible to express difficultviral proteins in a mammalian cell culture system where they aresecreted out of the cell. Additionally this fusion protein has furtherutility because it can be used to raise antibodies against the viralantigen in rats, mice or rabbits using the corresponding Scn ortholog.

FIG. 20 depicts the expression of a Scn fusion with an extracellularviral glycoprotein HIV gp120 and the corresponding SDS PAGE analysisaccording to one aspect of the present disclosure. The results in FIG.20 demonstrate that stabilized glycoproteins can be expressed accordingto one aspect of the present disclosure. Additionally this fusionprotein has further utility because it can be used to raise antibodiesagainst the viral antigen in rats, mice or rabbits using thecorresponding Scn ortholog.

FIG. 21 depicts the expression of a Scn fusion with a knottin protein,Imperatoxin, and the corresponding SDS PAGE analysis according to oneaspect of the present disclosure. The results in FIG. 21 show that theknottin protein, Imperatoxin, is stable even after separation from Scn.These results show that knottins can be expressed in a mammalian cellculture system where they are secreted out of the cell according to anaspect of the present disclosure. Although knottins are known to beincredibly difficult to secrete in a properly folded state, the resultsof FIG. 21 demonstrates that it is possible to do so using the presentlydescribed methods. Additionally, this fusion protein has further utilitybecause it can be used to raise antibodies against the viral antigen inrats, mice or rabbits using the corresponding Scn ortholog.

FIG. 22 depicts the expression of a Scn fusion with a small subdomain(i.e., Kringle domain) of the extracellular tyrosine kinase receptorROR1 and corresponding SDS PAGE analysis according to one aspect of thepresent disclosure. The results in FIG. 22 show that the Kringle domainfailed to express when alone, while it was successfully expressed aspart of a Scn fusion. These results show that the Kringle domain of theextracellular tyrosine kinase receptor ROR1 can be efficiently preparedaccording to one aspect of the present dislcosure. Additionally, thisfusion protein has further utility because it can be used to raiseantibodies against the viral antigen in rats, mice or rabbits using thecorresponding Scn ortholog.

FIG. 23 depicts the expression of a Scn fusion with heptameric andtrimeric subdomains and corresponding SDS PAGE analysis according to oneaspect of the present disclosure. As demonstrated in FIG. 23, thepresently described methods enable the expression of multimeric proteinconstructs in a mammalian cell culture system, where the proteins aresecreted out of the cell.

FIG. 24 depicts the expression of an ExFABP fusion with a knottin andcorresponding SDS PAGE analysis according to one aspect of the presentdisclosure. ExFABP is another functional Scn. According to the presentdisclosure, this construct can be used in a periplasmic bacterial systemto secret a variety of client proteins.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

SEQUENCES The following are DNA and/or amino acid sequences ofgenes of interest and constructs identified herein.Construction of parental construct for seamless cloning:IgK-SF-H6-GGS-Icn2C-GGS-ENLYFQ-GG-PARENTAL for Xho/Bamcut from pUC57 and ligation into pCVL SEQ ID NO: 1-GACTGAGTCGCCCGCTCGAGACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACTACAAGGACGAGCATCACCATCATCACCATGGTGGAAGCCAGGACTCCACCTCAGACCTGATCCCAGCCCCACCTCTGAGCAAGGTCCCTCTGCAGCAGAACTTCCAGGACAACCAATTCCAGGGGAAGTGGTATGTGGTAGGCCTGGCAGGGAATGCAATTCTCAGAGAAGACAAAGACCCGCAAAAGATGTATGCCACCATCTATGAGCTGAAAGAAGACAAGAGCTACAATGTCACCTCCGTCCTGTTTAGGAAAAAGAAGTGTGACTACTGGATCAGGACTTTTGTTCCAGGTTGCCAGCCCGGCGAGTTCACGCTGGGCAACATTAAGAGTTACCCTGGATTAACGAGTTACCTCGTCCGAGTGGTGAGCACCAACTACAACCAGCATGCTATGGTGTTCTTCAAGAAAGTTTCTCAAAACAGGGAGTACTTCAAGATCACCCTCTACGGGAGAACCAAGGAGCTGACTTCGGAACTAAAGGAGAACTTCATCCGCTTCTCCAAATCTCTGGGCCTCCCTGAAAACCACATCGTCTTCCCTGTCCCAATCGACCAGTGTATCGACGGCGGAGGTAGCGAAAACCTGTATTTTCAGGGAGGCGGCCGCTAAGGATCCCGGACCGCCTCTCC NotI cut is AACCTGTATTTTCAGGGAGGC - GCTAAGGATCCCGGACCGCCTCTCCFusion protein Sequences - original set of 10 - cloned intoNotI cut parent above by seamless cloning:IgK-SF-H6-GGS-Icn2C-GGS-ENLYFQ-GG-BubbleProtein SEQ ID NO: 2-GACTGAGTCGCCCGCTCGAGACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACTACAAGGACGAGCATCACCATCATCACCATGGTGGAAGCCAGGACTCCACCTCAGACCTGATCCCAGCCCCACCTCTGAGCAAGGTCCCTCTGCAGCAGAACTTCCAGGACAACCAATTCCAGGGGAAGTGGTATGTGGTAGGCCTGGCAGGGAATGCAATTCTCAGAGAAGACAAAGACCCGCAAAAGATGTATGCCACCATCTATGAGCTGAAAGAAGACAAGAGCTACAATGTCACCTCCGTCCTGTTTAGGAAAAAGAAGTGTGACTACTGGATCAGGACTTTTGTTCCAGGTTGCCAGCCCGGCGAGTTCACGCTGGGCAACATTAAGAGTTACCCTGGATTAACGAGTTACCTCGTCCGAGTGGTGAGCACCAACTACAACCAGCATGCTATGGTGTTCTTCAAGAAAGTTTCTCAAAACAGGGAGTACTTCAAGATCACCCTCTACGGGAGAACCAAGGAGCTGACTTCGGAACTAAAGGAGAACTTCATCCGCTTCTCCAAATCTCTGGGCCTCCCTGAAAACCACATCGTCTTCCCTGTCCCAATCGACCAGTGTATCGACGGCGGAGGTAGCGAAAACCTGTATTTTCAGGGAGGC GATACCTGCGGCAGCGGCTATAATGTGGATCAGCGTCGTACCAATAGCGGCTGCAAAGCGGGCAATGGCGATCGTCATTTTTGCGGCTGCGATCGTACCGGCGTGGTGGAATGCAAAGGCGGCAAATGGACCGAAGTGCAGGATTGCGGCAGCAGCAGCTGCAAAGGCACCAGCAATGGCGGCGCGACCTGC TAATGCTAAGGATCCC GGASEQ ID NO: 3-atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtM E T D T L L L W V L L L W V P G S T GgactacaaggacgagcatcaccatcatcaccatggtggaagccaggactccacctcagacD Y K D E H H H H H H G G S Q D S T S DctgatcccagccccacctctgagcaaggtccctctgcagcagaacttccaggacaaccaaL I P A P P L S K V P L Q Q N F Q D N QttccaggggaagtggtatgtggtaggcctggcagggaatgcaattctcagagaagacaaaF Q G K W Y V V G L A G N A I L R E D KgacccgcaaaagatgtatgccaccatctatgagctgaaagaagacaagagctacaatgtcD P Q K M Y A T I Y E L K E D K S Y N VacctccgtcctgtttaggaaaaagaagtgtgactactggatcaggacttttgttccaggtT S V L F R K K K C D Y W I R T F V P GtgccagcccggcgagttcacgctgggcaacattaagagttaccctggattaacgagttacC Q P G E F T L G N I K S Y P G L T S YctcgtccgagtggtgagcaccaactacaaccagcatgctatggtgttcttcaagaaagttL V R V V S T N Y N Q H A M V F F K K VtctcaaaacagggagtacttcaagatcaccctctacgggagaaccaaggagctgacttcgS Q N R E Y F K I T L Y G R T K E L T SgaactaaaggagaacttcatccgcttctccaaatctctgggcctccctgaaaaccacatcE L K E N F I R F S K S L G L P E N H IgtcttccctgtcccaatcgaccagtgtatcgacggcggaggtagcgaaaacctgtattttV F P V P I D Q C I D G G G S E N L Y FcagggaggcgatacctgcggcagcggctataatgtggatcagcgtcgtaccaatagcggcQ G G D T C G S G Y N V D Q R R T N S GtgcaaagcgggcaatggcgatcgtcatttttgcggctgcgatcgtaccggcgtggtggaaC K A G N G D R H F C G C D R T G V V EtgcaaaggcggcaaatggaccgaagtgcaggattgcggcagcagcagctgcaaaggcaccC K G G K W T E V Q D C G S S S C K G T agcaatggcggcgcgacctgcS N G G A T C IgK-SF-H6-GGS-Icn2C-GGS-ENLYFQ-GG-Attractin SEQ ID NO: 4-GACTGAGTCGCCCGCTCGAGACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACTACAAGGACGAGCATCACCATCATCACCATGGTGGAAGCCAGGACTCCACCTCAGACCTGATCCCAGCCCCACCTCTGAGCAAGGTCCCTCTGCAGCAGAACTTCCAGGACAACCAATTCCAGGGGAAGTGGTATGTGGTAGGCCTGGCAGGGAATGCAATTCTCAGAGAAGACAAAGACCCGCAAAAGATGTATGCCACCATCTATGAGCTGAAAGAAGACAAGAGCTACAATGTCACCTCCGTCCTGTTTAGGAAAAAGAAGTGTGACTACTGGATCAGGACTTTTGTTCCAGGTTGCCAGCCCGGCGAGTTCACGCTGGGCAACATTAAGAGTTACCCTGGATTAACGAGTTACCTCGTCCGAGTGGTGAGCACCAACTACAACCAGCATGCTATGGTGTTCTTCAAGAAAGTTTCTCAAAACAGGGAGTACTTCAAGATCACCCTCTACGGGAGAACCAAGGAGCTGACTTCGGAACTAAAGGAGAACTTCATCCGCTTCTCCAAATCTCTGGGCCTCCCTGAAAACCACATCGTCTTCCCTGTCCCAATCGACCAGTGTATCGACGGCGGAGGTAGCGAAAACCTGTATTTTCAGGGAGGC GATCAGAATTGCGATATTGGCAATATTACCAGCCAGTGCCAGATGCAGCATAAAAATTGCGAAGATGCGAATGGCTGCGATACCATTATTGAAGAATGCAAAACCAGCATGGTGGAACGTTGCCAGAATCAGGAATTTGAAAGCGCGGCGGGCAGCACCACCCTGGGCCCGCAG TAATGCTAAGGATCCCGGA SEQ ID NO: 5-atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtM E T D T L L L W V L L L W V P G S T GgactacaaggacgagcatcaccatcatcaccatggtggaagccaggactccacctcagacD Y K D E H H H H H H G G S Q D S T S DctgatcccagccccacctctgagcaaggtccctctgcagcagaacttccaggacaaccaaL I P A P P L S K V P L Q Q N F Q D N QttccaggggaagtggtatgtggtaggcctggcagggaatgcaattctcagagaagacaaaF Q G K W Y V V G L A G N A I L R E D KgacccgcaaaagatgtatgccaccatctatgagctgaaagaagacaagagctacaatgtcD P Q K M Y A T I Y E L K E D K S Y N VacctccgtcctgtttaggaaaaagaagtgtgactactggatcaggacttttgttccaggtT S V L F R K K K C D Y W I R T F V P GtgccagcccggcgagttcacgctgggcaacattaagagttaccctggattaacgagttacC Q P G E F T L G N I K S Y P G L T S YctcgtccgagtggtgagcaccaactacaaccagcatgctatggtgttcttcaagaaagttL V R V V S T N Y N Q H A M V F F K K VtctcaaaacagggagtacttcaagatcaccctctacgggagaaccaaggagctgacttcgS Q N R E Y F K I T L Y G R T K E L T SgaactaaaggagaacttcatccgcttctccaaatctctgggcctccctgaaaaccacatcE L K E N F I R F S K S L G L P E N H IgtcttccctgtcccaatcgaccagtgtatcgacggcggaggtagcgaaaacctgtattttV F P V P I D Q C I D G G G S E N L Y FcagggaggcgatcagaattgcgatattggcaatattaccagccagtgccagatgcagcatQ G G D Q N C D I G N I T S Q C Q M Q HaaaaattgcgaagatgcgaatggctgcgataccattattgaagaatgcaaaaccagcatgK N C E D A N G C D T I I E E C K T S MgtggaacgttgccagaatcaggaatttgaaagcgcggcgggcagcaccaccctgggcccgV E R C Q N Q E F E S A A G S T T L G P cag QIgK-SF-H6-GGS-Icn2C-GGS-ENLYFQ-GG-Hefutoxin SEQ ID NO: 6-GACTGAGTCGCCCGCTCGAGACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACTACAAGGACGAGCATCACCATCATCACCATGGTGGAAGCCAGGACTCCACCTCAGACCTGATCCCAGCCCCACCTCTGAGCAAGGTCCCTCTGCAGCAGAACTTCCAGGACAACCAATTCCAGGGGAAGTGGTATGTGGTAGGCCTGGCAGGGAATGCAATTCTCAGAGAAGACAAAGACCCGCAAAAGATGTATGCCACCATCTATGAGCTGAAAGAAGACAAGAGCTACAATGTCACCTCCGTCCTGTTTAGGAAAAAGAAGTGTGACTACTGGATCAGGACTTTTGTTCCAGGTTGCCAGCCCGGCGAGTTCACGCTGGGCAACATTAAGAGTTACCCTGGATTAACGAGTTACCTCGTCCGAGTGGTGAGCACCAACTACAACCAGCATGCTATGGTGTTCTTCAAGAAAGTTTCTCAAAACAGGGAGTACTTCAAGATCACCCTCTACGGGAGAACCAAGGAGCTGACTTCGGAACTAAAGGAGAACTTCATCCGCTTCTCCAAATCTCTGGGCCTCCCTGAAAACCACATCGTCTTCCCTGTCCCAATCGACCAGTGTATCGACGGCGGAGGTAGCGAAAACCTGTATTTTCAGGGAGGC GGCCATGCGTGCTATCGTAATTGCTGGCGTGAAGGCAATGATGAAGAAACCTGCAAAGAACGTTGC TAATGCTAAGGATCCCGGACCGCC SEQ ID NO: 7-atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtM E T D T L L L W V L L L W V P G S T GgactacaaggacgagcatcaccatcatcaccatggtggaagccaggactccacctcagacD Y K D E H H H H H H G G S Q D S T S DctgatcccagccccacctctgagcaaggtccctctgcagcagaacttccaggacaaccaaL I P A P P L S K V P L Q Q N F Q D N QttccaggggaagtggtatgtggtaggcctggcagggaatgcaattctcagagaagacaaaF Q G K W Y V V G L A G N A I L R E D KgacccgcaaaagatgtatgccaccatctatgagctgaaagaagacaagagctacaatgtcD P Q K M Y A T I Y E L K E D K S Y N VacctccgtcctgtttaggaaaaagaagtgtgactactggatcaggacttttgttccaggtT S V L F R K K K C D Y W I R T F V P GtgccagcccggcgagttcacgctgggcaacattaagagttaccctggattaacgagttacC Q P G E F T L G N I K S Y P G L T S YctcgtccgagtggtgagcaccaactacaaccagcatgctatggtgttcttcaagaaagttL V R V V S T N Y N Q H A M V F F K K VtctcaaaacagggagtacttcaagatcaccctctacgggagaaccaaggagctgacttcgS Q N R E Y F K I T L Y G R T K E L T SgaactaaaggagaacttcatccgcttctccaaatctctgggcctccctgaaaaccacatcE L K E N F I R F S K S L G L P E N H IgtcttccctgtcccaatcgaccagtgtatcgacggcggaggtagcgaaaacctgtattttV F P V P I D Q C I D G G G S E N L Y FcagggaggcggccatgcgtgctatcgtaattgctggcgtgaaggcaatgatgaagaaaccQ G G G H A C Y R N C W R E G N D E E T tgcaaagaacgttgc C K E R CIgK-SF-H6-GGS-Icn2C-GGS-ENLYFQ-GG-Hanatoxin SEQ ID NO: 8-GACTGAGTCGCCCGCTCGAGACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACTACAAGGACGAGCATCACCATCATCACCATGGTGGAAGCCAGGACTCCACCTCAGACCTGATCCCAGCCCCACCTCTGAGCAAGGTCCCTCTGCAGCAGAACTTCCAGGACAACCAATTCCAGGGGAAGTGGTATGTGGTAGGCCTGGCAGGGAATGCAATTCTCAGAGAAGACAAAGACCCGCAAAAGATGTATGCCACCATCTATGAGCTGAAAGAAGACAAGAGCTACAATGTCACCTCCGTCCTGTTTAGGAAAAAGAAGTGTGACTACTGGATCAGGACTTTTGTTCCAGGTTGCCAGCCCGGCGAGTTCACGCTGGGCAACATTAAGAGTTACCCTGGATTAACGAGTTACCTCGTCCGAGTGGTGAGCACCAACTACAACCAGCATGCTATGGTGTTCTTCAAGAAAGTTTCTCAAAACAGGGAGTACTTCAAGATCACCCTCTACGGGAGAACCAAGGAGCTGACTTCGGAACTAAAGGAGAACTTCATCCGCTTCTCCAAATCTCTGGGCCTCCCTGAAAACCACATCGTCTTCCCTGTCCCAATCGACCAGTGTATCGACGGCGGAGGTAGCGAAAACCTGTATTTTCAGGGAGGC GAATGCCGTTATCTGTTTGGCGGCTGCAAAACCACCAGCGATTGCTGCAAACATCTGGGCTGCAAATTTCGTGATAAATATTGCGCGTGGGATT TTACCTTTAGCTAATGCTAAGGATCCCGGA SEQ ID NO: 9-atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtM E T D T L L L W V L L L W V P G S T GgactacaaggacgagcatcaccatcatcaccatggtggaagccaggactccacctcagacD Y K D E H H H H H H G G S Q D S T S DctgatcccagccccacctctgagcaaggtccctctgcagcagaacttccaggacaaccaaL I P A P P L S K V P L Q Q N F Q D N QttccaggggaagtggtatgtggtaggcctggcagggaatgcaattctcagagaagacaaaF Q G K W Y V V G L A G N A I L R E D KgacccgcaaaagatgtatgccaccatctatgagctgaaagaagacaagagctacaatgtcD P Q K M Y A T I Y E L K E D K S Y N VacctccgtcctgtttaggaaaaagaagtgtgactactggatcaggacttttgttccaggtT S V L F R K K K C D Y W I R T F V P GtgccagcccggcgagttcacgctgggcaacattaagagttaccctggattaacgagttacC Q P G E F T L G N I K S Y P G L T S YctcgtccgagtggtgagcaccaactacaaccagcatgctatggtgttcttcaagaaagttL V R V V S T N Y N Q H A M V F F K K VtctcaaaacagggagtacttcaagatcaccctctacgggagaaccaaggagctgacttcgS Q N R E Y F K I T L Y G R T K E L T SgaactaaaggagaacttcatccgcttctccaaatctctgggcctccctgaaaaccacatcE L K E N F I R F S K S L G L P E N H IgtcttccctgtcccaatcgaccagtgtatcgacggcggaggtagcgaaaacctgtattttV F P V P I D Q C I D G G G S E N L Y FcagggaggcgaatgccgttatctgtttggcggctgcaaaaccaccagcgattgctgcaaaQ G G E C R Y L F G G C K T T S D C C KcatctgggctgcaaatttcgtgataaatattgcgcgtgggattttacctttagcH L G C K F R D K Y C A W D F T F SIgK-SF-H6-GGS-Icn2C-GGS-ENLYFQ-GG-ChymotrypsinInhibitor SEQ ID NO: 10-GACTGAGTCGCCCGCTCGAGACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACTACAAGGACGAGCATCACCATCATCACCATGGTGGAAGCCAGGACTCCACCTCAGACCTGATCCCAGCCCCACCTCTGAGCAAGGTCCCTCTGCAGCAGAACTTCCAGGACAACCAATTCCAGGGGAAGTGGTATGTGGTAGGCCTGGCAGGGAATGCAATTCTCAGAGAAGACAAAGACCCGCAAAAGATGTATGCCACCATCTATGAGCTGAAAGAAGACAAGAGCTACAATGTCACCTCCGTCCTGTTTAGGAAAAAGAAGTGTGACTACTGGATCAGGACTTTTGTTCCAGGTTGCCAGCCCGGCGAGTTCACGCTGGGCAACATTAAGAGTTACCCTGGATTAACGAGTTACCTCGTCCGAGTGGTGAGCACCAACTACAACCAGCATGCTATGGTGTTCTTCAAGAAAGTTTCTCAAAACAGGGAGTACTTCAAGATCACCCTCTACGGGAGAACCAAGGAGCTGACTTCGGAACTAAAGGAGAACTTCATCCGCTTCTCCAAATCTCTGGGCCTCCCTGAAAACCACATCGTCTTCCCTGTCCCAATCGACCAGTGTATCGACGGCGGAGGTAGCGAAAACCTGTATTTTCAGGGAGGC GAAATTAGCTGCGAACCGGGCAAAACCTTTAAAGATAAATGCAATACCTGCCGTTGCGGCGCGGATGGCAAAAGCGCGGCGTGCACCCTGAAAGCGTGCCCGAATCAG TAATGCTAAGGATCCCGGA SEQ ID NO: 11-atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtM E T D T L L L W V L L L W V P G S T GgactacaaggacgagcatcaccatcatcaccatggtggaagccaggactccacctcagacD Y K D E H H H H H H G G S Q D S T S DctgatcccagccccacctctgagcaaggtccctctgcagcagaacttccaggacaaccaaL I P A P P L S K V P L Q Q N F Q D N QttccaggggaagtggtatgtggtaggcctggcagggaatgcaattctcagagaagacaaaF Q G K W Y V V G L A G N A I L R E D KgacccgcaaaagatgtatgccaccatctatgagctgaaagaagacaagagctacaatgtcD P Q K M Y A T I Y E L K E D K S Y N VacctccgtcctgtttaggaaaaagaagtgtgactactggatcaggacttttgttccaggtT S V L F R K K K C D Y W I R T F V P GtgccagcccggcgagttcacgctgggcaacattaagagttaccctggattaacgagttacC Q P G E F T L G N I K S Y P G L T S YctcgtccgagtggtgagcaccaactacaaccagcatgctatggtgttcttcaagaaagttL V R V V S T N Y N Q H A M V F F K K VtctcaaaacagggagtacttcaagatcaccctctacgggagaaccaaggagctgacttcgS Q N R E Y F K I T L Y G R T K E L T SgaactaaaggagaacttcatccgcttctccaaatctctgggcctccctgaaaaccacatcE L K E N F I R F S K S L G L P E N H IgtcttccctgtcccaatcgaccagtgtatcgacggcggaggtagcgaaaacctgtattttV F P V P I D Q C I D G G G S E N L Y FcagggaggcgaaattagctgcgaaccgggcaaaacctttaaagataaatgcaatacctgcQ G G E I S C E P G K T F K D K C N T CcgttgcggcgcggatggcaaaagcgcggcgtgcaccctgaaagcgtgcccgaatcagR C G A D G K S A A C T L K A C P N QIgK-SF-H6-GGS-Icn2C-GGS-ENLYFQ-GG-ToxinK SEQ ID NO: 12-GACTGAGTCGCCCGCTCGAGACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACTACAAGGACGAGCATCACCATCATCACCATGGTGGAAGCCAGGACTCCACCTCAGACCTGATCCCAGCCCCACCTCTGAGCAAGGTCCCTCTGCAGCAGAACTTCCAGGACAACCAATTCCAGGGGAAGTGGTATGTGGTAGGCCTGGCAGGGAATGCAATTCTCAGAGAAGACAAAGACCCGCAAAAGATGTATGCCACCATCTATGAGCTGAAAGAAGACAAGAGCTACAATGTCACCTCCGTCCTGTTTAGGAAAAAGAAGTGTGACTACTGGATCAGGACTTTTGTTCCAGGTTGCCAGCCCGGCGAGTTCACGCTGGGCAACATTAAGAGTTACCCTGGATTAACGAGTTACCTCGTCCGAGTGGTGAGCACCAACTACAACCAGCATGCTATGGTGTTCTTCAAGAAAGTTTCTCAAAACAGGGAGTACTTCAAGATCACCCTCTACGGGAGAACCAAGGAGCTGACTTCGGAACTAAAGGAGAACTTCATCCGCTTCTCCAAATCTCTGGGCCTCCCTGAAAACCACATCGTCTTCCCTGTCCCAATCGACCAGTGTATCGACGGCGGAGGTAGCGAAAACCTGTATTTTCAGGGAGGC GTGTGCCGTGATTGGTTTAAAGAAACCGCGTGCCGTCATGCGAAAAGCCTGGGCAATTGCCGTACCAGCCAGAAATATCGTGCGAATTGCGCGAAAACCTGCGAACTGTGC TAATGCTAAGGATCCCGGA SEQ ID NO: 13-atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtM E T D T L L L W V L L L W V P G S T GgactacaaggacgagcatcaccatcatcaccatggtggaagccaggactccacctcagacD Y K D E H H H H H H G G S Q D S T S DctgatcccagccccacctctgagcaaggtccctctgcagcagaacttccaggacaaccaaL I P A P P L S K V P L Q Q N F Q D N QttccaggggaagtggtatgtggtaggcctggcagggaatgcaattctcagagaagacaaaF Q G K W Y V V G L A G N A I L R E D KgacccgcaaaagatgtatgccaccatctatgagctgaaagaagacaagagctacaatgtcD P Q K M Y A T I Y E L K E D K S Y N VacctccgtcctgtttaggaaaaagaagtgtgactactggatcaggacttttgttccaggtT S V L F R K K K C D Y W I R T F V P GtgccagcccggcgagttcacgctgggcaacattaagagttaccctggattaacgagttacC Q P G E F T L G N I K S Y P G L T S YctcgtccgagtggtgagcaccaactacaaccagcatgctatggtgttcttcaagaaagttL V R V V S T N Y N Q H A M V F F K K VtctcaaaacagggagtacttcaagatcaccctctacgggagaaccaaggagctgacttcgS Q N R E Y F K I T L Y G R T K E L T SgaactaaaggagaacttcatccgcttctccaaatctctgggcctccctgaaaaccacatcE L K E N F I R F S K S L G L P E N H IgtcttccctgtcccaatcgaccagtgtatcgacggcggaggtagcgaaaacctgtattttV F P V P I D Q C I D G G G S E N L Y FcagggaggcgtgtgccgtgattggtttaaagaaaccgcgtgccgtcatgcgaaaagcctgQ G G V C R D W F K E T A C R H A K S LggcaattgccgtaccagccagaaatatcgtgcgaattgcgcgaaaacctgcgaactgtgcG N C R T S Q K Y R A N C A K T C E L CIgK-SF-H6-GGS-Icn2C-GGS-ENLYFQ-GG-EGFepiregulinCore SEQ ID NO: 14-GACTGAGTCGCCCGCTCGAGACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACTACAAGGACGAGCATCACCATCATCACCATGGTGGAAGCCAGGACTCCACCTCAGACCTGATCCCAGCCCCACCTCTGAGCAAGGTCCCTCTGCAGCAGAACTTCCAGGACAACCAATTCCAGGGGAAGTGGTATGTGGTAGGCCTGGCAGGGAATGCAATTCTCAGAGAAGACAAAGACCCGCAAAAGATGTATGCCACCATCTATGAGCTGAAAGAAGACAAGAGCTACAATGTCACCTCCGTCCTGTTTAGGAAAAAGAAGTGTGACTACTGGATCAGGACTTTTGTTCCAGGTTGCCAGCCCGGCGAGTTCACGCTGGGCAACATTAAGAGTTACCCTGGATTAACGAGTTACCTCGTCCGAGTGGTGAGCACCAACTACAACCAGCATGCTATGGTGTTCTTCAAGAAAGTTTCTCAAAACAGGGAGTACTTCAAGATCACCCTCTACGGGAGAACCAAGGAGCTGACTTCGGAACTAAAGGAGAACTTCATCCGCTTCTCCAAATCTCTGGGCCTCCCTGAAAACCACATCGTCTTCCCTGTCCCAATCGACCAGTGTATCGACGGCGGAGGTAGCGAAAACCTGTATTTTCAGGGAGGC GTGAGCATTACCAAATGCAGCAGCGATATGAATGGCTATTGCCTGCATGGCCAGTGCATTTATCTGGTGGATATGAGCCAGAATTATTGCCGTTGCGAAGTGGGCTATACCGGCGTGCGTTGCGAACATTTTTTTCTG TAATGCTAAGGATCCC GGASEQ ID NO: 15-atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtM E T D T L L L W V L L L W V P G S T GgactacaaggacgagcatcaccatcatcaccatggtggaagccaggactccacctcagacD Y K D E H H H H H H G G S Q D S T S DctgatcccagccccacctctgagcaaggtccctctgcagcagaacttccaggacaaccaaL I P A P P L S K V P L Q Q N F Q D N QttccaggggaagtggtatgtggtaggcctggcagggaatgcaattctcagagaagacaaaF Q G K W Y V V G L A G N A I L R E D KgacccgcaaaagatgtatgccaccatctatgagctgaaagaagacaagagctacaatgtcD P Q K M Y A T I Y E L K E D K S Y N VacctccgtcctgtttaggaaaaagaagtgtgactactggatcaggacttttgttccaggtT S V L F R K K K C D Y W I R T F V P GtgccagcccggcgagttcacgctgggcaacattaagagttaccctggattaacgagttacC Q P G E F T L G N I K S Y P G L T S YctcgtccgagtggtgagcaccaactacaaccagcatgctatggtgttcttcaagaaagttL V R V V S T N Y N Q H A M V F F K K VtctcaaaacagggagtacttcaagatcaccctctacgggagaaccaaggagctgacttcgS Q N R E Y F K I T L Y G R T K E L T SgaactaaaggagaacttcatccgcttctccaaatctctgggcctccctgaaaaccacatcE L K E N F I R F S K S L G L P E N H IgtcttccctgtcccaatcgaccagtgtatcgacggcggaggtagcgaaaacctgtattttV F P V P I D Q C I D G G G S E N L Y FcagggaggcgtgagcattaccaaatgcagcagcgatatgaatggctattgcctgcatggcQ G G V S I T K C S S D M N G Y C L H GcagtgcatttatctggtggatatgagccagaattattgccgttgcgaagtgggctataccQ C I Y L V D M S Q N Y C R C E V G Y T ggcgtgcgttgcgaacatttttttctgG V R C E H F F L IgK-SF-H6-GGS-Icn2C-GGS-ENLYFQ-GG-CirculinSEQ ID NO: 16-GACTGAGTCGCCCGCTCGAGACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACTACAAGGACGAGCATCACCATCATCACCATGGTGGAAGCCAGGACTCCACCTCAGACCTGATCCCAGCCCCACCTCTGAGCAAGGTCCCTCTGCAGCAGAACTTCCAGGACAACCAATTCCAGGGGAAGTGGTATGTGGTAGGCCTGGCAGGGAATGCAATTCTCAGAGAAGACAAAGACCCGCAAAAGATGTATGCCACCATCTATGAGCTGAAAGAAGACAAGAGCTACAATGTCACCTCCGTCCTGTTTAGGAAAAAGAAGTGTGACTACTGGATCAGGACTTTTGTTCCAGGTTGCCAGCCCGGCGAGTTCACGCTGGGCAACATTAAGAGTTACCCTGGATTAACGAGTTACCTCGTCCGAGTGGTGAGCACCAACTACAACCAGCATGCTATGGTGTTCTTCAAGAAAGTTTCTCAAAACAGGGAGTACTTCAAGATCACCCTCTACGGGAGAACCAAGGAGCTGACTTCGGAACTAAAGGAGAACTTCATCCGCTTCTCCAAATCTCTGGGCCTCCCTGAAAACCACATCGTCTTCCCTGTCCCAATCGACCAGTGTATCGACGGCGGAGGTAGCGAAAACCTGTATTTTCAGGGAGGC GGCATTCCGTGCGGCGAAAGCTGCGTGTGGATTCCGTGCATTAGCGCGGCGCTGGGCTGCAGCTGCAAAAATAAAGTGTGCTATCGTAAT TAATGCTAAGGATCCCGGA SEQ ID NO: 17-atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtM E T D T L L L W V L L L W V P G S T GgactacaaggacgagcatcaccatcatcaccatggtggaagccaggactccacctcagacD Y K D E H H H H H H G G S Q D S T S DctgatcccagccccacctctgagcaaggtccctctgcagcagaacttccaggacaaccaaL I P A P P L S K V P L Q Q N F Q D N QttccaggggaagtggtatgtggtaggcctggcagggaatgcaattctcagagaagacaaaF Q G K W Y V V G L A G N A I L R E D KgacccgcaaaagatgtatgccaccatctatgagctgaaagaagacaagagctacaatgtcD P Q K M Y A T I Y E L K E D K S Y N VacctccgtcctgtttaggaaaaagaagtgtgactactggatcaggacttttgttccaggtT S V L F R K K K C D Y W I R T F V P GtgccagcccggcgagttcacgctgggcaacattaagagttaccctggattaacgagttacC Q P G E F T L G N I K S Y P G L T S YctcgtccgagtggtgagcaccaactacaaccagcatgctatggtgttcttcaagaaagttL V R V V S T N Y N Q H A M V F F K K VtctcaaaacagggagtacttcaagatcaccctctacgggagaaccaaggagctgacttcgS Q N R E Y F K I T L Y G R T K E L T SgaactaaaggagaacttcatccgcttctccaaatctctgggcctccctgaaaaccacatcE L K E N F I R F S K S L G L P E N H IgtcttccctgtcccaatcgaccagtgtatcgacggcggaggtagcgaaaacctgtattttV F P V P I D Q C I D G G G S E N L Y FcagggaggcggcattccgtgcggcgaaagctgcgtgtggattccgtgcattagcgcggcgQ G G G I P C G E S C V W I P C I S A Actgggctgcagctgcaaaaataaagtgtgctatcgtaat L G C S C K N K V C Y R NIgK-SF-H6-GGS-Icn2C-GGS-ENLYFQ-GG-Brazzein SEQ ID NO: 18-GACTGAGTCGCCCGCTCGAGACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACTACAAGGACGAGCATCACCATCATCACCATGGTGGAAGCCAGGACTCCACCTCAGACCTGATCCCAGCCCCACCTCTGAGCAAGGTCCCTCTGCAGCAGAACTTCCAGGACAACCAATTCCAGGGGAAGTGGTATGTGGTAGGCCTGGCAGGGAATGCAATTCTCAGAGAAGACAAAGACCCGCAAAAGATGTATGCCACCATCTATGAGCTGAAAGAAGACAAGAGCTACAATGTCACCTCCGTCCTGTTTAGGAAAAAGAAGTGTGACTACTGGATCAGGACTTTTGTTCCAGGTTGCCAGCCCGGCGAGTTCACGCTGGGCAACATTAAGAGTTACCCTGGATTAACGAGTTACCTCGTCCGAGTGGTGAGCACCAACTACAACCAGCATGCTATGGTGTTCTTCAAGAAAGTTTCTCAAAACAGGGAGTACTTCAAGATCACCCTCTACGGGAGAACCAAGGAGCTGACTTCGGAACTAAAGGAGAACTTCATCCGCTTCTCCAAATCTCTGGGCCTCCCTGAAAACCACATCGTCTTCCCTGTCCCAATCGACCAGTGTATCGACGGCGGAGGTAGCGAAAACCTGTATTTTCAGGGAGGC CAGGATAAATGCAAAAAAGTGTATGAAAATTATCCGGTGAGCAAATGCCAGCTGGCGAATCAGTGCAATTATGATTGCAAACTGGATAAACATGCGCGTAGCGGCGAATGCTTTTATGATGAAAAACGTAATCTGCAGTGCATTTGCGATTAT TGCGAATATTAATGCTAAGGATCCCGGA SEQ ID NO: 19-atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtM E T D T L L L W V L L L W V P G S T GgactacaaggacgagcatcaccatcatcaccatggtggaagccaggactccacctcagacD Y K D E H H H H H H G G S Q D S T S DctgatcccagccccacctctgagcaaggtccctctgcagcagaacttccaggacaaccaaL I P A P P L S K V P L Q Q N F Q D N QttccaggggaagtggtatgtggtaggcctggcagggaatgcaattctcagagaagacaaaF Q G K W Y V V G L A G N A I L R E D KgacccgcaaaagatgtatgccaccatctatgagctgaaagaagacaagagctacaatgtcD P Q K M Y A T I Y E L K E D K S Y N VacctccgtcctgtttaggaaaaagaagtgtgactactggatcaggacttttgttccaggtT S V L F R K K K C D Y W I R T F V P GtgccagcccggcgagttcacgctgggcaacattaagagttaccctggattaacgagttacC Q P G E F T L G N I K S Y P G L T S YctcgtccgagtggtgagcaccaactacaaccagcatgctatggtgttcttcaagaaagttL V R V V S T N Y N Q H A M V F F K K VtctcaaaacagggagtacttcaagatcaccctctacgggagaaccaaggagctgacttcgS Q N R E Y F K I T L Y G R T K E L T SgaactaaaggagaacttcatccgcttctccaaatctctgggcctccctgaaaaccacatcE L K E N F I R F S K S L G L P E N H IgtcttccctgtcccaatcgaccagtgtatcgacggcggaggtagcgaaaacctgtattttV F P V P I D Q C I D G G G S E N L Y FcagggaggccaggataaatgcaaaaaagtgtatgaaaattatccggtgagcaaatgccagQ G G Q D K C K K V Y E N Y P V S K C QctggcgaatcagtgcaattatgattgcaaactggataaacatgcgcgtagcggcgaatgcL A N Q C N Y D C K L D K H A R S G E CttttatgatgaaaaacgtaatctgcagtgcatttgcgattattgcgaatatF Y D E K R N L Q C I C D Y C E Y SEQ ID NO: 20-atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtM E T D T L L L W V L L L W V P G S T GgactacaaggacgagcatcaccatcatcaccatggtggaagccaggactccacctcagacD Y K D E H H H H H H G G S Q D S T S DctgatcccagccccacctctgagcaaggtccctctgcagcagaacttccaggacaaccaaL I P A P P L S K V P L Q Q N F Q D N QttccaggggaagtggtatgtggtaggcctggcagggaatgcaattctcagagaagacaaaF Q G K W Y V V G L A G N A I L R E D KgacccgcaaaagatgtatgccaccatctatgagctgaaagaagacaagagctacaatgtcD P Q K M Y A T I Y E L K E D K S Y N VacctccgtcctgtttaggaaaaagaagtgtgactactggatcaggacttttgttccaggtT S V L F R K K K C D Y W I R T F V P GtgccagcccggcgagttcacgctgggcaacattaagagttaccctggattaacgagttacC Q P G E F T L G N I K S Y P G L T S YctcgtccgagtggtgagcaccaactacaaccagcatgctatggtgttcttcaagaaagttL V R V V S T N Y N Q H A M V F F K K VtctcaaaacagggagtacttcaagatcaccctctacgggagaaccaaggagctgacttcgS Q N R E Y F K I T L Y G R T K E L T SgaactaaaggagaacttcatccgcttctccaaatctctgggcctccctgaaaaccacatcE L K E N F I R F S K S L G L P E N H IgtcttccctgtcccaatcgaccagtgtatcgacggcggaggtagcgaaaacctgtattttV F P V P I D Q C I D G G G S E N L Y FcagggaggcatgtgcatgccgtgctttaccaccgatcatcagatggcgcgtaaatgcgatQ G G M C M P C F T T D H Q M A R K C DgattgctgcggcggcaaaggccgtggcaaatgctatggcccgcagtgcctgtgccgtD C C G G K G R G K C Y G P Q C L C RConstruction of parental construct for BamH1/NotI cloning:IgK-SF-H6-GGS-Icn2C-GGS-ENLYFQ-GS-PARENTAL SEQ ID NO: 21-GACTGAGTCGCCCGCTCGAGACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACTACAAGGACGAGCATCACCATCATCACCATGGTGGAAGCCAGGACTCCACCTCAGACCTGATCCCAGCCCCACCTCTGAGCAAGGTCCCTCTGCAGCAGAACTTCCAGGACAACCAATTCCAGGGGAAGTGGTATGTGGTAGGCCTGGCAGGGAATGCAATTCTCAGAGAAGACAAAGACCCGCAAAAGATGTATGCCACCATCTATGAGCTGAAAGAAGACAAGAGCTACAATGTCACCTCCGTCCTGTTTAGGAAAAAGAAGTGTGACTACTGGATCAGGACTTTTGTTCCAGGTTGCCAGCCCGGCGAGTTCACGCTGGGCAACATTAAGAGTTACCCTGGATTAACGAGTTACCTCGTCCGAGTGGTGAGCACCAACTACAACCAGCATGCTATGGTGTTCTTCAAGAAAGTTTCTCAAAACAGGGAGTACTTCAAGATCACCCTCTACGGGAGAACCAAGGAGCTGACTTCGGAACTAAAGGAGAACTTCATCCGCTTCTCCAAATCTCTGGGCCTCCCTGAAAACCACATCGTCTTCCCTGTCCCAATCGACCAGTGTATCGACGGCGGAGGTAGCGAAAACCTGTATTTTCAGGGATCCTAATGTTGGCCATGATGTTAGGCGGCCGCTAAGGA TCCCGGABamHI site: GGATCC NotI site: GCGGCCGC A BamHI site adds “GS”before a knottin. This construct can be used for cloning libraries.Construction of parental construct for furin cleavage,BamHI/NotI cloning can include an idealized furin cutsite is RARYKRS-RARYKRGS can be used for a Bam HI site.IgK-SF-H6-GGS-Icn2C-GGS-furin-GS-PARENTAL SEQ ID NO: 22-GACTGAGTCGCCCGCTCGAGACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACTACAAGGACGAGCATCACCATCATCACCATGGTGGAAGCCAGGACTCCACCTCAGACCTGATCCCAGCCCCACCTCTGAGCAAGGTCCCTCTGCAGCAGAACTTCCAGGACAACCAATTCCAGGGGAAGTGGTATGTGGTAGGCCTGGCAGGGAATGCAATTCTCAGAGAAGACAAAGACCCGCAAAAGATGTATGCCACCATCTATGAGCTGAAAGAAGACAAGAGCTACAATGTCACCTCCGTCCTGTTTAGGAAAAAGAAGTGTGACTACTGGATCAGGACTTTTGTTCCAGGTTGCCAGCCCGGCGAGTTCACGCTGGGCAACATTAAGAGTTACCCTGGATTAACGAGTTACCTCGTCCGAGTGGTGAGCACCAACTACAACCAGCATGCTATGGTGTTCTTCAAGAAAGTTTCTCAAAACAGGGAGTACTTCAAGATCACCCTCTACGGGAGAACCAAGGAGCTGACTTCGGAACTAAAGGAGAACTTCATCCGCTTCTCCAAATCTCTGGGCCTCCCTGAAAACCACATCGTCTTCCCTGTCCCAATCGACCAGTGTATCGACGGCGGAGGTAGCcgcgcgcgctataaacgcGGATCCTAATGTTGGCCATGATGTTAGGCGGCCGCTAAGGATCCC GGAIgK-SF-H6-GGS-Icn2C-GGS-ENLYFQ-GS-MIDKINE SEQ ID NO: 23-GACTGAGTCGCCCGCTCGAGACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACTACAAGGACGAGCATCACCATCATCACCATGGTGGAAGCCAGGACTCCACCTCAGACCTGATCCCAGCCCCACCTCTGAGCAAGGTCCCTCTGCAGCAGAACTTCCAGGACAACCAATTCCAGGGGAAGTGGTATGTGGTAGGCCTGGCAGGGAATGCAATTCTCAGAGAAGACAAAGACCCGCAAAAGATGTATGCCACCATCTATGAGCTGAAAGAAGACAAGAGCTACAATGTCACCTCCGTCCTGTTTAGGAAAAAGAAGTGTGACTACTGGATCAGGACTTTTGTTCCAGGTTGCCAGCCCGGCGAGTTCACGCTGGGCAACATTAAGAGTTACCCTGGATTAACGAGTTACCTCGTCCGAGTGGTGAGCACCAACTACAACCAGCATGCTATGGTGTTCTTCAAGAAAGTTTCTCAAAACAGGGAGTACTTCAAGATCACCCTCTACGGGAGAACCAAGGAGCTGACTTCGGAACTAAAGGAGAACTTCATCCGCTTCTCCAAATCTCTGGGCCTCCCTGAAAACCACATCGTCTTCCCTGTCCCAATCGACCAGTGTATCGACGGCGGAGGTAGCGAAAACCTGTATTTTCAGAGCGATTGCAAATATAAATTTGAAAACTGGGGCGCGTGCGATGGCGGCACCGGCACCAAAGTGCGCCAGGGCACCCTGAAAAAAGCGCGCTATAACGCGCAGTGCCAGGAAACCATTCGCGTGACCAAACCGTGCTAAT GCT GGATCCCGGACCGCCTCT CCSEQ ID NO: 24-atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtM E T D T L L L W V L L L W V P G S T GgactacaaggacgagcatcaccatcatcaccatggtggaagccaggactccacctcagacD Y K D E H H H H H H G G S Q D S T S DctgatcccagccccacctctgagcaaggtccctctgcagcagaacttccaggacaaccaaL I P A P P L S K V P L Q Q N F Q D N QttccaggggaagtggtatgtggtaggcctggcagggaatgcaattctcagagaagacaaaF Q G K W Y V V G L A G N A I L R E D KgacccgcaaaagatgtatgccaccatctatgagctgaaagaagacaagagctacaatgtcD P Q K M Y A T I Y E L K E D K S Y N VacctccgtcctgtttaggaaaaagaagtgtgactactggatcaggacttttgttccaggtT S V L F R K K K C D Y W I R T F V P GtgccagcccggcgagttcacgctgggcaacattaagagttaccctggattaacgagttacC Q P G E F T L G N I K S Y P G L T S YctcgtccgagtggtgagcaccaactacaaccagcatgctatggtgttcttcaagaaagttL V R V V S T N Y N Q H A M V F F K K VtctcaaaacagggagtacttcaagatcaccctctacgggagaaccaaggagctgacttcgS Q N R E Y F K I T L Y G R T K E L T SgaactaaaggagaacttcatccgcttctccaaatctctgggcctccctgaaaaccacatcE L K E N F I R F S K S L G L P E N H IgtcttccctgtcccaatcgaccagtgtatcgacggcggaggtagcgaaaacctgtattttV F P V P I D Q C I D G G G S E N L Y FcagagcgattgcaaatataaatttgaaaactggggcgcgtgcgatggcggcaccggcaccQ S D C K Y K F E N W G A C D G G T G TaaagtgcgccagggcaccctgaaaaaagcgcgctataacgcgcagtgccaggaaaccattK V R Q G T L K K A R Y N A Q C Q E T I cgcgtgaccaaaccgtgc R V T K P CIgK-SF-H6-GGS-Icn2C-GGS-ENLYFQ-GG-Violacin A SEQ ID NO: 25-GACTGAGTCGCCCGCTCGAGACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACTACAAGGACGAGCATCACCATCATCACCATGGTGGAAGCCAGGACTCCACCTCAGACCTGATCCCAGCCCCACCTCTGAGCAAGGTCCCTCTGCAGCAGAACTTCCAGGACAACCAATTCCAGGGGAAGTGGTATGTGGTAGGCCTGGCAGGGAATGCAATTCTCAGAGAAGACAAAGACCCGCAAAAGATGTATGCCACCATCTATGAGCTGAAAGAAGACAAGAGCTACAATGTCACCTCCGTCCTGTTTAGGAAAAAGAAGTGTGACTACTGGATCAGGACTTTTGTTCCAGGTTGCCAGCCCGGCGAGTTCACGCTGGGCAACATTAAGAGTTACCCTGGATTAACGAGTTACCTCGTCCGAGTGGTGAGCACCAACTACAACCAGCATGCTATGGTGTTCTTCAAGAAAGTTTCTCAAAACAGGGAGTACTTCAAGATCACCCTCTACGGGAGAACCAAGGAGCTGACTTCGGAACTAAAGGAGAACTTCATCCGCTTCTCCAAATCTCTGGGCCTCCCTGAAAACCACATCGTCTTCCCTGTCCCAATCGACCAGTGTATCGACGGCGGAGGTAGCGAAAACCTGTATTTTCAGGGAGGCAGCGCCATCAGCTGCGGCGAGACCTGCTTCAAGTTCAAGTGCTACACCCCCAGATGCAGCTGCAGCTACCCCGTGTGCAAGTAAGCTAAGGATC CCGGACCGCCSEQ ID NO: 26-atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtM E T D T L L L W V L L L W V P G S T GgactacaaggacgagcatcaccatcatcaccatggtggaagccaggactccacctcagacD Y K D E H H H H H H G G S Q D S T S DctgatcccagccccacctctgagcaaggtccctctgcagcagaacttccaggacaaccaaL I P A P P L S K V P L Q Q N F Q D N QttccaggggaagtggtatgtggtaggcctggcagggaatgcaattctcagagaagacaaaF Q G K W Y V V G L A G N A I L R E D KgacccgcaaaagatgtatgccaccatctatgagctgaaagaagacaagagctacaatgtcD P Q K M Y A T I Y E L K E D K S Y N VacctccgtcctgtttaggaaaaagaagtgtgactactggatcaggacttttgttccaggtT S V L F R K K K C D Y W I R T F V P GtgccagcccggcgagttcacgctgggcaacattaagagttaccctggattaacgagttacC Q P G E F T L G N I K S Y P G L T S YctcgtccgagtggtgagcaccaactacaaccagcatgctatggtgttcttcaagaaagttL V R V V S T N Y N Q H A M V F F K K VtctcaaaacagggagtacttcaagatcaccctctacgggagaaccaaggagctgacttcgS Q N R E Y F K I T L Y G R T K E L T SgaactaaaggagaacttcatccgcttctccaaatctctgggcctccctgaaaaccacatcE L K E N F I R F S K S L G L P E N H IgtcttccctgtcccaatcgaccagtgtatcgacggcggaggtagcgaaaacctgtattttV F P V P I D Q C I D G G G S E N L Y FcagggaggcagcgccatcagctgcggcgagacctgcttcaagttcaagtgctacacccccQ G G S A I S C G E T C F K F K C Y T P agatgcagctgcagctaccccgtgtgcaagR C S C S Y P V C K IgK-SF-H6-GGS-Icn2C-GGS-ENLYFQ-GG-Lambda ToxinSEQ ID NO: 27-GACTGAGTCGCCCGCTCGAGACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACTACAAGGACGAGCATCACCATCATCACCATGGTGGAAGCCAGGACTCCACCTCAGACCTGATCCCAGCCCCACCTCTGAGCAAGGTCCCTCTGCAGCAGAACTTCCAGGACAACCAATTCCAGGGGAAGTGGTATGTGGTAGGCCTGGCAGGGAATGCAATTCTCAGAGAAGACAAAGACCCGCAAAAGATGTATGCCACCATCTATGAGCTGAAAGAAGACAAGAGCTACAATGTCACCTCCGTCCTGTTTAGGAAAAAGAAGTGTGACTACTGGATCAGGACTTTTGTTCCAGGTTGCCAGCCCGGCGAGTTCACGCTGGGCAACATTAAGAGTTACCCTGGATTAACGAGTTACCTCGTCCGAGTGGTGAGCACCAACTACAACCAGCATGCTATGGTGTTCTTCAAGAAAGTTTCTCAAAACAGGGAGTACTTCAAGATCACCCTCTACGGGAGAACCAAGGAGCTGACTTCGGAACTAAAGGAGAACTTCATCCGCTTCTCCAAATCTCTGGGCCTCCCTGAAAACCACATCGTCTTCCCTGTCCCAATCGACCAGTGTATCGACGGCGGAGGTAGCGAAAACCTGTATTTTCAGGGAGGCGTGTGCTGCGGCTACAAGCTGTGCCACCCCTGCTAAGCTAAGGATCCCGGACC SEQ ID NO: 28-atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtM E T D T L L L W V L L L W V P G S T GgactacaaggacgagcatcaccatcatcaccatggtggaagccaggactccacctcagacD Y K D E H H H H H H G G S Q D S T S DctgatcccagccccacctctgagcaaggtccctctgcagcagaacttccaggacaaccaaL I P A P P L S K V P L Q Q N F Q D N QttccaggggaagtggtatgtggtaggcctggcagggaatgcaattctcagagaagacaaaF Q G K W Y V V G L A G N A I L R E D KgacccgcaaaagatgtatgccaccatctatgagctgaaagaagacaagagctacaatgtcD P Q K M Y A T I Y E L K E D K S Y N VacctccgtcctgtttaggaaaaagaagtgtgactactggatcaggacttttgttccaggtT S V L F R K K K C D Y W I R T F V P GtgccagcccggcgagttcacgctgggcaacattaagagttaccctggattaacgagttacC Q P G E F T L G N I K S Y P G L T S YctcgtccgagtggtgagcaccaactacaaccagcatgctatggtgttcttcaagaaagttL V R V V S T N Y N Q H A M V F F K K VtctcaaaacagggagtacttcaagatcaccctctacgggagaaccaaggagctgacttcgS Q N R E Y F K I T L Y G R T K E L T SgaactaaaggagaacttcatccgcttctccaaatctctgggcctccctgaaaaccacatcE L K E N F I R F S K S L G L P E N H IgtcttccctgtcccaatcgaccagtgtatcgacggcggaggtagcgaaaacctgtattttV F P V P I D Q C I D G G G S E N L Y Fcagggaggcgtgtgctgcggctacaagctgtgccacccctgc Q G G V C C G Y K L C H P CIgK-SF-H6-GGS-Icn2C-GGS-ENLYFQ-GG-Lambda Toxin NG SEQ ID NO: 29-GACTGAGTCGCCCGCTCGAGACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACTACAAGGACGAGCATCACCATCATCACCATGGTGGAAGCCAGGACTCCACCTCAGACCTGATCCCAGCCCCACCTCTGAGCAAGGTCCCTCTGCAGCAGAACTTCCAGGACAACCAATTCCAGGGGAAGTGGTATGTGGTAGGCCTGGCAGGGAATGCAATTCTCAGAGAAGACAAAGACCCGCAAAAGATGTATGCCACCATCTATGAGCTGAAAGAAGACAAGAGCTACAATGTCACCTCCGTCCTGTTTAGGAAAAAGAAGTGTGACTACTGGATCAGGACTTTTGTTCCAGGTTGCCAGCCCGGCGAGTTCACGCTGGGCAACATTAAGAGTTACCCTGGATTAACGAGTTACCTCGTCCGAGTGGTGAGCACCAACTACAACCAGCATGCTATGGTGTTCTTCAAGAAAGTTTCTCAAAACAGGGAGTACTTCAAGATCACCCTCTACGGGAGAACCAAGGAGCTGACTTCGGAACTAAAGGAGAACTTCATCCGCTTCTCCAAATCTCTGGGCCTCCCTGAAAACCACATCGTCTTCCCTGTCCCAATCGACCAGTGTATCGACGGCGGAGGTAGCGAAAACCTGTATTTTCAGGGAGGCAACGGCGTGTGCTGCGGCTACAAGCTGTGCCACCCCTGCTAAGCTAAGGATCCCGGACC SEQ ID NO: 30-atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtM E T D T L L L W V L L L W V P G S T GgactacaaggacgagcatcaccatcatcaccatggtggaagccaggactccacctcagacD Y K D E H H H H H H G G S Q D S T S DctgatcccagccccacctctgagcaaggtccctctgcagcagaacttccaggacaaccaaL I P A P P L S K V P L Q Q N F Q D N QttccaggggaagtggtatgtggtaggcctggcagggaatgcaattctcagagaagacaaaF Q G K W Y V V G L A G N A I L R E D KgacccgcaaaagatgtatgccaccatctatgagctgaaagaagacaagagctacaatgtcD P Q K M Y A T I Y E L K E D K S Y N VacctccgtcctgtttaggaaaaagaagtgtgactactggatcaggacttttgttccaggtT S V L F R K K K C D Y W I R T F V P GtgccagcccggcgagttcacgctgggcaacattaagagttaccctggattaacgagttacC Q P G E F T L G N I K S Y P G L T S YctcgtccgagtggtgagcaccaactacaaccagcatgctatggtgttcttcaagaaagttL V R V V S T N Y N Q H A M V F F K K VtctcaaaacagggagtacttcaagatcaccctctacgggagaaccaaggagctgacttcgS Q N R E Y F K I T L Y G R T K E L T SgaactaaaggagaacttcatccgcttctccaaatctctgggcctccctgaaaaccacatcE L K E N F I R F S K S L G L P E N H IgtcttccctgtcccaatcgaccagtgtatcgacggcggaggtagcgaaaacctgtattttV F P V P I D Q C I D G G G S E N L Y FcagggaggcaacggcgtgtgctgcggctacaagctgtgccacccctgcQ G G N G V C C G Y K L C H P CIgK-SF-H6-GGS-Icn2C-GGS-ENLYFQ-GG-Potato Carboxypeptidase InhibitorSEQ ID NO: 31-GACTGAGTCGCCCGCTCGAGACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACTACAAGGACGAGCATCACCATCATCACCATGGTGGAAGCCAGGACTCCACCTCAGACCTGATCCCAGCCCCACCTCTGAGCAAGGTCCCTCTGCAGCAGAACTTCCAGGACAACCAATTCCAGGGGAAGTGGTATGTGGTAGGCCTGGCAGGGAATGCAATTCTCAGAGAAGACAAAGACCCGCAAAAGATGTATGCCACCATCTATGAGCTGAAAGAAGACAAGAGCTACAATGTCACCTCCGTCCTGTTTAGGAAAAAGAAGTGTGACTACTGGATCAGGACTTTTGTTCCAGGTTGCCAGCCCGGCGAGTTCACGCTGGGCAACATTAAGAGTTACCCTGGATTAACGAGTTACCTCGTCCGAGTGGTGAGCACCAACTACAACCAGCATGCTATGGTGTTCTTCAAGAAAGTTTCTCAAAACAGGGAGTACTTCAAGATCACCCTCTACGGGAGAACCAAGGAGCTGACTTCGGAACTAAAGGAGAACTTCATCCGCTTCTCCAAATCTCTGGGCCTCCCTGAAAACCACATCGTCTTCCCTGTCCCAATCGACCAGTGTATCGACGGCGGAGGTAGCGAAAACCTGTATTTTCAGGGAGGCcagcagcatgcggatccgatttgcaacaaaccgtgcaaaacccatgatgattgcagcggcgcgtggttttgccaggcgtgctggaacagcgcgcgcacctgcggcccgtatgtgggcTAATGCTAAGGATCCCGGACCG SEQ ID NO: 32-atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtM E T D T L L L W V L L L W V P G S T GgactacaaggacgagcatcaccatcatcaccatggtggaagccaggactccacctcagacD Y K D E H H H H H H G G S Q D S T S DctgatcccagccccacctctgagcaaggtccctctgcagcagaacttccaggacaaccaaL I P A P P L S K V P L Q Q N F Q D N QttccaggggaagtggtatgtggtaggcctggcagggaatgcaattctcagagaagacaaaF Q G K W Y V V G L A G N A I L R E D KgacccgcaaaagatgtatgccaccatctatgagctgaaagaagacaagagctacaatgtcD P Q K M Y A T I Y E L K E D K S Y N VacctccgtcctgtttaggaaaaagaagtgtgactactggatcaggacttttgttccaggtT S V L F R K K K C D Y W I R T F V P GtgccagcccggcgagttcacgctgggcaacattaagagttaccctggattaacgagttacC Q P G E F T L G N I K S Y P G L T S YctcgtccgagtggtgagcaccaactacaaccagcatgctatggtgttcttcaagaaagttL V R V V S T N Y N Q H A M V F F K K VtctcaaaacagggagtacttcaagatcaccctctacgggagaaccaaggagctgacttcgS Q N R E Y F K I T L Y G R T K E L T SgaactaaaggagaacttcatccgcttctccaaatctctgggcctccctgaaaaccacatcE L K E N F I R F S K S L G L P E N H IgtcttccctgtcccaatcgaccagtgtatcgacggcggaggtagcgaaaacctgtattttV F P V P I D Q C I D G G G S E N L Y FcagggaggccagcagcatgcggatccgatttgcaacaaaccgtgcaaaacccatgatgatQ G G Q Q H A D P I C N K P C K T H D DtgcagcggcgcgtggttttgccaggcgtgctggaacagcgcgcgcacctgcggcccgtatC S G A W F C Q A C W N S A R T C G P Y gtgggctaa V GAnti-CD3-STa bispecificThe anti-CD3 is an OKT3 variant from the C-terminus ofpatent 7635462. It is supposed to work as either N- or C- terminal.SEQ ID NO: 33-GSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNVVYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSNSSNYCCELCCNPACTGCYThis is designed to be dropped into the Bam/Not cut library vector as aTEV-cleavable siderocalin fusion: SEQ ID NO: 34-ACCTGTATTTTCAGGGATCCgatattaaactgcagcagagcggcgcggaactggcgcgcccgggcgcgagcgtgaaaatgagctgcaaaaccagcggctatacctttacccgctataccatgcattgggtgaaacagcgcccgggccagggcctggaatggattggctatattaacccgagccgcggctataccaactataaccagaaatttaaagataaagcgaccctgaccaccgataaaagcagcagcaccgcgtatatgcagctgagcagcctgaccagcgaagatagcgcggtgtattattgcgcgcgctattatgatgatcattattgcctggattattggggccagggcaccaccctgaccgtgagcagcgtggaaggcggcagcggcggcagcggcggcagcggcggcagcggcggcgtggatgatattcagctgacccagagcccggcgattatgagcgcgagcccgggcgaaaaagtgaccatgacctgccgcgcgagcagcagcgtgagctatatgaactggtatcagcagaaaagcggcaccagcccgaaacgctggatttatgataccagcaaagtggcgagcggcgtgccgtatcgctttagcggcagcggcagcggcaccagctatagcctgaccattagcagcatggaagcggaagatgcggcgacctattattgccagcagtggagcagcaacccgctgacctttggcgcgggcaccaaactggaactgaaaggcggcggcggcagcaacagcagcaactattgctgcgaactgtgctgcaacccggcgtgcaccggctgctatTAATGCGGCCGCTCATCACCATTAATC Parental cloning construct 1 for downstream fusions:IgK-sFLAG-H6-GGS-humanScn-GGS-ENLYFQ-GG-NotI SEQ ID NO: 35-CTCGAGACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACTACAAGGACGAGCATCACCATCATCACCATGGTGGAAGCCAGGACTCCACCTCAGACCTGATCCCAGCCCCACCTCTGAGCAAGGTCCCTCTGCAGCAGAACTTCCAGGACAACCAATTCCAGGGGAAGTGGTATGTGGTAGGCCTGGCAGGGAATGCAATTCTCAGAGAAGACAAAGACCCGCAAAAGATGTATGCCACCATCTATGAGCTGAAAGAAGACAAGAGCTACAATGTCACCTCCGTCCTGTTTAGGAAAAAGAAGTGTGACTACTGGATCAGGACTTTTGTTCCAGGTTGCCAGCCCGGCGAGTTCACGCTGGGCAACATTAAGAGTTACCCTGGATTAACGAGTTACCTCGTCCGAGTGGTGAGCACCAACTACAACCAGCATGCTATGGTGTTCTTCAAGAAAGTTTCTCAAAACAGGGAGTACTTCAAGATCACCCTCTACGGGAGAACCAAGGAGCTGACTTCGGAACTAAAGGAGAACTTCATCCGCTTCTCCAAATCTCTGGGCCTCCCTGAAAACCACATCGTCTTCCCTGTCCCAATCGACCAGTGTATCGACGGCGGAGGTAGCGAAAACCTGTATTTTCAGGGAGGCGGCCGCParental cloning construct 2 for downstream fusions:IgK-sFLAG-H6-GGS-humanScnC87S-GGS-ENLYFQ-GS-STUFFER SEQ ID NO: 36-

ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACTACAAGGACGAGCATCACCATCATCACCATGGTGGAAGCCAGGACTCCACCTCAGACCTGATCCCAGCCCCACCTCTGAGCAAGGTCCCTCTGCAGCAGAACTTCCAGGACAACCAATTCCAGGGGAAGTGGTATGTGGTAGGCCTGGCAGGGAATGCAATTCTCAGAGAAGACAAAGACCCGCAAAAGATGTATGCCACCATCTATGAGCTGAAAGAAGACAAGAGCTACAATGTCACCTCCGTCCTGTTTAGGAAAAAGAAGTGTGACTACTGGATCAGGACTTTTGTTCCAGGTTCCCAGCCCGGCGAGTTCACGCTGGGCAACATTAAGAGTTACCCTGGATTAACGAGTTACCTCGTCCGAGTGGTGAGCACCAACTACAACCAGCATGCTATGGTGTTCTTCAAGAAAGTTTCTCAAAACAGGGAGTACTTCAAGATCACCCTCTACGGGAGAACCAAGGAGCTGACTTCGGAACTAAAGGAGAACTTCATCCGCTTCTCCAAATCTCTGGGCCTCCCTGAAAACCACATCGTCTTCCCTGTCCCAATCGACCAGTGTATCGACGGCGGAGGTAGCGAAAACCTGTATTTTCAGGGATCCATGTACGGTCTTAAGGGACCCGACATTTACAAAGGAGTTTACCAATTTAAGTCAGTGGAGTTTGATATGTCACATCTGAACCTGACCATGCCCAACGCATGTTCAGCCAACAACTCCCACCATTACATCAGTATGGGGACTTCTGGACTAGAATTGACCTTCACCAATGATTCCATCATCAGTCACAACTTTTGCAATCTGACCTCTGCCTTCAACAAAAAGACCTTTGACCACACACTCATGAGTATAGTTTCGAGCCTACACCTCAGTATCAGAGGGAACTCCAACTATAAGGCAGTATCCTGCGACTTCAACAATGGCATAACCATCCAATACAACTTGACATTCTCAGATCGACAAAGTGCTCAGAGCCAGTGTAGAACCTTCAGAGGTAGAGTCCTAGATATGTTTAGAACTGCCTTCGGGGGGAAATACATGAGGAGTGGCTGGGGCTGGACAGGCTCAGATGGCAAGACCACCTGGTGTAGCCAGACGAGTTACCAATACCTGATTATACAAAATAGAACCTGGGAAAACCACTGCACATATGCAGGTCCTTTTGGGATGTCCAGGATTCTCCTTTCCCAAGAGAAGACTAAGTTCTTCACTAGGAGACTGGTGCCCAGGGGCAGCGGCCTGAACGACATCTTCGAGGCCCAGAAGATCGAGTGGCACGAGTAATGCGGCCGCTCATCACCATTAATCATCACCATTAATCGGACCG Parental cloning construct 3 for light chain fusions:LightChain-GRGGSGGS-humanScnC87S SEQ ID NO: 37-CTCGAGACCATGGATTTCCAGGTGCAGATTTTTAGCTTTCTGCTGATTTCCGCTTCCGTGATTATGAGCCGAGGCGACATTGTGATGACCCAGGCAGCTCCTAGCGTGCCAGTCACCCCAGGAGAGTCAGTGAGCATCTCCTGCAGAAGTACTAAGTCACTGCTGCACAGCAACGGCAATACCTACCTGTATTGGTTCCTGCAGAGACCTGGGCAGTCCCCACAGAGGCTGATCTACTATATGAGTAACCTGGCATCAGGAGTGCCTGACAGGTTCAGCGGACGAGGCAGCGGCACTGATTTTACCCTGCGGATTTCTAGAGTGGAGGCAGAAGACGCCGGCGTCTACTATTGCATGCAGAGTCTGGAGTACCCTTATACTTTCGGCGGGGGAACCAAACTGGAAATCAAGAGGGCCGATGCCGCTCCAACCGTGTCCATTTTTCCCCCTAGCTCCGAGCAGCTGACATCTGGCGGGGCTAGTGTGGTCTGTTTCCTGAACAATTTTTACCCAAAGGACATCAACGTGAAATGGAAGATTGATGGAAGTGAAAGGCAGAACGGCGTCCTGAATTCATGGACAGACCAGGATAGCAAAGACTCCACTTATTCTATGTCTAGTACCCTGACACTGACTAAGGATGAGTACGAACGCCACAATTCTTATACATGCGAGGCAACTCATAAAACCTCTACAAGTCCCATCGTGAAGAGCTTTAACCGAAATGAATGCGGCCGCGGAGGCTCCGGAGGCTCCCAGGACTCAACAAGCGATCTGATTCCAGCCCCACCCCTGAGCAAAGTGCCCCTGCAGCAGAACTTCCAGGACAATCAGTTTCAGGGCAAGTGGTACGTGGTCGGGCTGGCTGGAAACGCAATCCTGCGGGAGGACAAAGATCCCCAGAAGATGTACGCCACTATCTACGAGCTGAAAGAAGACAAGTCATACAATGTGACCAGCGTCCTGTTCCGCAAGAAAAAGTGTGATTATTGGATCAGAACATTCGTGCCCGGCTCCCAGCCTGGGGAGTTTACTCTGGGGAATATTAAGTCCTACCCTGGACTGACCTCTTATCTGGTGCGAGTGGTCTCTACAAACTACAATCAGCATGCTATGGTGTTCTTTAAAAAGGTCAGCCAGAACCGGGAGTACTTTAAAATCACCCTGTATGGCAGAACCAAAGAACTGACAAGCGAGCTGAAGGAAAATTTCATTCGCTTTTCCAAGTCTCTGGGGCTGCCAGAGAATCATATTGTGTTCCCAGTCCCCATTGACCAGTGTATTGACGGGTGAGGATCC Super stable ScnC87ShumanScn-I8C-N39C-C87S SEQ ID NO: 38-CTCGAGATGCCCCTGGGCCTGCTGTGGCTGGGCCTGGCCCTGCTGGGCGCCCTGCACGCCCAGGCCCAGGACTCCACCTCAGACCTGTGTCCAGCCCCACCTCTGAGCAAGGTCCCTCTGCAGCAGAACTTCCAGGACAACCAATTCCAGGGGAAGTGGTATGTGGTAGGCCTGGCAGGGTGTGCAATTCTCAGAGAAGACAAAGACCCGCAAAAGATGTATGCCACCATCTATGAGCTGAAAGAAGACAAGAGCTACAATGTCACCTCCGTCCTGTTTAGGAAAAAGAAGTGTGACTACTGGATCAGGACTTTTGTTCCAGGTTCCCAGCCGGGCGAGTTCACGCTGGGCAACATTAAGAGTTACCCTGGATTAACGAGTTACCTCGTCCGAGTGGTGAGCACCAACTACAACCAGCATGCTATGGTGTTCTTCAAGAAAGTTTCTCAAAACAGGGAGTACTTCAAGATCACCCTCTACGGGAGAACCAAGGAGCTGACTTCGGAACTAAAGGAGAACTTCATCCGCTTCTCCAAATCTCTGGGCCTCCCTGAAAACCACATCGTCTTCCCTGTCCCAATCGACCAGTGTATCGACGGCGGATCC Homosapiens SEQ ID NO: 39-QDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGCQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDG PantroglodytesSEQ ID NO: 40-QDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGRQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELQENFIRFSKSLGLPENHIVFPVPIDQCIDG CanisfamiliarisSEQ ID NO: 41-QDSTPSLIPAPPPLKVPLQPDFQHDQFQGKWYVTGIAGNILKKEGHGQLKMYTTTYELKDDQSYNVTSTLLRNERCDYWNRDFVPSFQPGQFSLGDIQLYPGVQSYLVQVVATNYNQYALVYFRKVYKSQEYFKITLYGRTKELPLELKKEFIRFAKSIGLTEDHIIFPVPIDQCIDE BostaurusSEQ ID NO: 42-RSSSSRLLRAPPLSRIPLQPNFQADQFQGKWYTVGVAGNAIKKEEQDPLKMYSSNYELKEDGSYNVTSILLKDDLCDYWIRTFVPSSQPGQFTLGNIKSYRGIRSYTVRVVNTDYNQFAIVYFKKVQRKKTYFKITLYGRTKELTPEVRENFINFAKSLGLTDDHIVFTVPIDRCIDDQ MusmusculusSEQ ID NO: 43-QDSTQNLIPAPSLLTVPLQPDFRSDQFRGRWYVVGLAGNAVQKKTEGSFTMYSTIYELQENNSYNVTSILVRDQDQGCRYWIRTFVPSSRAGQFTLGNMHRYPQVQSYNVQVATTDYNQFAMVFFRKTSENKQYFKITLYGRTKELSPELKERFTRFAKSLGLKDDNIIFSVPTDQCIDN RattusnorvegicusSEQ ID NO: 44-QDSTQNLIPAPPLISVPLQPGFWTERFQGRWFVVGLAANAVQKERQSRFTMYSTIYELQEDNSYNVTSILVRGQGCRYWIRTFVPSSRPGQFTLGNIHSYPQIQSYDVQVADTDYDQFAMVFFQKTSENKQYFKVTLYGRTKGLSDELKERFVSFAKSLGLKDNNIVFSVPTDQCIDN MacacamulattaSEQ ID NO: 45-QDSSSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLSGNAVGRKDEAPLKMYATIYELKEDKSYNVTSILFRKEKCDYWIRTFVPGSQPGEFTLGNIQNHPGLTSYVVRVVSTNYKQYAMVFFKKVSQNKEYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFSVPIDQCING TursiopstruncatusSEQ ID NO: 46-QDSTPNLIPAPPLFRVPLQPNFQPDQFQGKWYIVGLAGNAFKKEKQGQFKMYATTYELKEDRSYNVTSALLRGKTQRCDHWIRTFVPSSRPGQFTLGNIKGFPGVQSYTVRVATTNYNQFAIVYFKKVYKNQEYFKTTLYGRTKELTPQLKENFIHFAKSLGLTDEYILFPVPIDKCIDDQ GorillagorillaSEQ ID NO: 47-QDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFREKAQKCDYWIRTFVPGSQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDG ProcaviacapensisSEQ ID NO: 48-QEPTPTLIPAPPLSSIPLKPNFHNDKFQGKWYVVGVAGNAITKEKDPSLMYTTTYELRDDGSYNVTSTQFREKINCTHWTRTFVPTSQPGQFSLGNIDKYPHLSSYTVRVTATNYNYFAIVYFKKVSKNQEYFKTTLYKRIKKLTHGLKKHFIQFAKSLGLPDNHITFLVPTDRCIDDA CallithrixjacchusSEQ ID NO: 49-QDSPSPLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAIRREDQDSLKMYATIYELKEDKSYNVTSVLFRKAQKCDYWIRTFVPSSRPGEFKLGNIESHPGLTSYIVRVVNTDYKQHAMVFFMKASHNRKYFKVTLYGRTKELTSDLKENFTSFSKSLGLTENHIIFPVPIDQCIDG MicrocebusmurinusSEQ ID NO: 50-QDSKEKLIPAPPLLRVPLQPDFQDDQFRETSWPRGSKMKETPAGSRDAGTGWATTYELKDHSYNVTSTLLRQNGKCDYWIRTFVLTSQPGQFALGNINRYPGIQSYTVRVVTTNYNQFAIVFFKKVSENKEYFKTTLYGRTKELPPELKENFIRFAKSLGLTEDHIIYPVPIDQCIDD SEQ ID NO: 51-QDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAIRREDKDSQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGSQPGEFTLGNTKGYPGLTSYLVRVVSTNYNQYAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPAPIDQCIDG OchotonaprincepsSEQ ID NO: 52-QELTTDLIPVPSLRKIHVQKNFQSDQFQGKWYVVGLAGNNIHNSDQEHQQMYSTTYELKEDGSYNVTSTLLRQRNQQCDHWIRTFVPGSKLGHFNLGNIKSYPTLKSYLIRVVTTDYNQFAIVFFRKVYKNNKKFFKIVLYGRTKELSPELRGRFTSFAKTLGLTDNHIVFPAPIGQCIDD LoxodontaafricanaSEQ ID NO: 53-QTHSPTLIPAPPLLRVPLQPDFQDDKFQGKWYVIGLAGNAVEKKEQGQFKMYTTTYELKEDGSYNVTSTLLQEDGKCSYWIRTFVPSFQPGQFNLGNIKNFPGLQSYTVRVTATNYNQFAIVFFKKVSKNGEYFKTTLYGRTKELTPELKERFIRFAKSLGLSDHIIFPVPIDRCIDD OryctolaguscuniculusSEQ ID NO: 54-QDPTPKLIPAPSLRRVPLQRNFQDEQFQGKWYVVGLAGNAVQKREEGQEPMYSTTYELNEDRSFNVTSTLLRDQRCDHWIRTFVPTSRPGQYNLGNIKSYPGVKNYIVRVVATDYSQYAMMFFRKGSRNKQFFKTTLYGRTKELSPELRERFTRFAKSLGLPDDRIVFPTPIDQCIDDMurine Scn construct for downstream viral fusions:IgK-H6-murineScn-StrepII-GGGGS-E7.16gi|29468134|gb|AAO85409.1|AF402678_2 E7 [HPV16] SEQ ID NO: 55-CTCGAGACCATGGAGACCGACACGCTCTTGTTGTGGGTTCTCTTGTTGTGGGTGCCTGGGTCTACAGGCGACCACCACCATCATCACCACCTCGTTCCTAGAGGCAGCCAGGATAGTACCCAGAATCTTATCCCAGCACCATCTTTGCTCACAGTACCATTGCAACCCGACTTTCGGTCTGATCAATTTCGGGGACGCTGGTACGTGGTTGGACTGGCCGGCAATGCTGTACAGAAAAAAACAGAGGGCAGTTTCACCATGTACTCAACAATCTATGAGCTCCAAGAGAATAATAGTTACAACGTTACCTCCATCTTGGTGAGGGACCAGGATCAGGGATGTCGCTACTGGATTCGGACATTCGTACCAAGTTCTCGGGCCGGTCAGTTTACTCTGGGCAACATGCACAGGTATCCCCAAGTTCAATCTTACAACGTGCAGGTGGCGACTACCGACTACAACCAATTCGCTATGGTGTTCTTCCGCAAAACAAGCGAGAACAAGCAGTATTTTAAAATCACTCTGTACGGTAGAACTAAGGAGCTGAGCCCTGAACTTAAGGAGCGGTTCACCAGATTCGCTAAGTCCCTGGGACTGAAGGATGATAATATAATCTTTTCCGTCCCCACCGATCAGTGTATCGATAATTCAGCTTGGTCACATCCCCAGTTCGAGAAAGGAGGCGGTGGATCCATGCATGGCGACACTCCGACCCTGCACGAATACATGCTGGACCTGCAGCCCGAAACCACTGACCTGTATTGTTACGAGCAACTCAACGATTCTAGCGAGGAGGAGGACGAGATCGATGGACCGGCAGGCCAGGCCGAACCAGACCGCGCACATTATAACATTGTTACCTTCTGTTGCAAATGTGATTCAACTCTTAGACTTTGTGTCCAGAGTACACACGTGGACATCCGCACCCTGGAAGATCTGCTGATGGGAACTCTGGGTATCGTGTGTCCTATATGTAGCCAGAAACCCTGACGGACCG SEQ ID NO: 56-METDTLLLWVLLLWVPGSTGDHHHHHHLVPRGSQDSTQNLIPAPSLLTVPLQPDFRSDQFRGRWYVVGLAGNAVQKKTEGSFTMYSTIYELQENNSYNVTSILVRDQDQGCRYWIRTFVPSSRAGQFTLGNMHRYPQVQSYNVQVATTDYNQFAMVFFRKTSENKQYFKITLYGRTKELSPELKERFTRFAKSLGLKDDNIIFSVPTDQCIDN

IgK-H6-murineScn-StrepII-GGGGS-E6.16gi|4927720|gb|AAD33252.1|AF125673_1 E6 [HPV16] SEQ ID NO: 57-CTCGAGACCATGGAGACCGACACGCTCTTGTTGTGGGTTCTCTTGTTGTGGGTGCCTGGGTCTACAGGCGACCACCACCATCATCACCACCTCGTTCCTAGAGGCAGCCAGGATAGTACCCAGAATCTTATCCCAGCACCATCTTTGCTCACAGTACCATTGCAACCCGACTTTCGGTCTGATCAATTTCGGGGACGCTGGTACGTGGTTGGACTGGCCGGCAATGCTGTACAGAAAAAAACAGAGGGCAGTTTCACCATGTACTCAACAATCTATGAGCTCCAAGAGAATAATAGTTACAACGTTACCTCCATCTTGGTGAGGGACCAGGATCAGGGATGTCGCTACTGGATTCGGACATTCGTACCAAGTTCTCGGGCCGGTCAGTTTACTCTGGGCAACATGCACAGGTATCCCCAAGTTCAATCTTACAACGTGCAGGTGGCGACTACCGACTACAACCAATTCGCTATGGTGTTCTTCCGCAAAACAAGCGAGAACAAGCAGTATTTTAAAATCACTCTGTACGGTAGAACTAAGGAGCTGAGCCCTGAACTTAAGGAGCGGTTCACCAGATTCGCTAAGTCCCTGGGACTGAAGGATGATAATATAATCTTTTCCGTCCCCACCGATCAGTGTATCGATAATTCAGCTTGGTCACATCCCCAGTTCGAGAAAGGAGGCGGTGGATCCATGCACCAGAAGAGAACCGCCATGTTCCAGGAcCCaCAAGAGCGGCCCCGGAAACTGCCCCAACTGTGCACTGAATTGCAGACCACCATCCACGACATCATTTTGGAATGTGTCTACTGTAAGCAGCAGCTCCTCAGGCGAGAGGTGTATGACTTCGCCTTCCGGGATTTGTGTATTGTCTACAGGGATGGTAATCCCTATGCCGTTTGTGATAAGTGCCTGAAATTTTATAGCAAGATCAGCGAGTACCGACATTACTGTTACAGCGTTTATGGAACAACATTGGAGCAGCAGTACAACAAACCTCTTTGCGACCTCCTGATTCGCTGCATCAACTGCCAGAAGCCCCTGTGCCCCGAAGAGAAACAAAGGCATTTGGATAAGAAGCAGAGGTTCCACAACATCCGCGGTCGCTGGACGGGGCGCTGCATGAGTTGCTGCAGGAGTTCCCGCACTCGGCGCGAGACCCAACTCTGACGGACCGCCTCTCCCTCCCIgK-H6-murineScn-StrepII-GGGGS-E6.18 gi|30172005|gb|AAP20594.1|E6 protein [HPV18] SEQ ID NO: 58-CTCGAGACCATGGAGACCGACACGCTCTTGTTGTGGGTTCTCTTGTTGTGGGTGCCTGGGTCTACAGGCGACCACCACCATCATCACCACCTCGTTCCTAGAGGCAGCCAGGATAGTACCCAGAATCTTATCCCAGCACCATCTTTGCTCACAGTACCATTGCAACCCGACTTTCGGTCTGATCAATTTCGGGGACGCTGGTACGTGGTTGGACTGGCCGGCAATGCTGTACAGAAAAAAACAGAGGGCAGTTTCACCATGTACTCAACAATCTATGAGCTCCAAGAGAATAATAGTTACAACGTTACCTCCATCTTGGTGAGGGACCAGGATCAGGGATGTCGCTACTGGATTCGGACATTCGTACCAAGTTCTCGGGCCGGTCAGTTTACTCTGGGCAACATGCACAGGTATCCCCAAGTTCAATCTTACAACGTGCAGGTGGCGACTACCGACTACAACCAATTCGCTATGGTGTTCTTCCGCAAAACAAGCGAGAACAAGCAGTATTTTAAAATCACTCTGTACGGTAGAACTAAGGAGCTGAGCCCTGAACTTAAGGAGCGGTTCACCAGATTCGCTAAGTCCCTGGGACTGAAGGATGATAATATAATCTTTTCCGTCCCCACCGATCAGTGTATCGATAATTCAGCTTGGTCACATCCCCAGTTCGAGAAAGGAGGCGGTGGATCCATGGCCAGATTTGAAGACCCCACAAGGCGCCCCTATAAACTGCCGGATCTTTGCACCGAACTGAATACTAGCCTGCAAGATATTGAGATTACCTGCGTGTACTGTAAAACGGTGCTCGAATTGACCGAGGTTTTTGAGTTCGCATTCAAGGACCTGTTTGTTGTATATCGCGATTCCATCCCGCACGCAGCTTGCCATAAATGCATTGACTTTTACTCCCGGATACGCGAGCTGCGACACTATAGTGATAGCGTGTACGGCGATACACTTGAGAAGCTTACCAACACCGGTCTGTACAATCTTCTGATTCGGTGTTTGAGGTGCCAGAAGCCGCTCAACCCAGCTGAGAAACTGCGGCATCTGAACGAAAAAAGAAGATTCCACAACATTGCTGGCCACTACAGGGGCCAGTGCCATTCTTGTTGTAATAGAGCAAGGCAGGAGCGGCTGCAACGGCGGCGCGAGACCCAGGTATGACGGACCGCCTCTCCCTCCCIgK-H6-murineScn-StrepII-GGGGS-E7.18 gi|285804409|gb|ADC35717.1|E7 [HPV 18] SEQ ID NO: 59-CTCGAGACCATGGAGACCGACACGCTCTTGTTGTGGGTTCTCTTGTTGTGGGTGCCTGGGTCTACAGGCGACCACCACCATCATCACCACCTCGTTCCTAGAGGCAGCCAGGATAGTACCCAGAATCTTATCCCAGCACCATCTTTGCTCACAGTACCATTGCAACCCGACTTTCGGTCTGATCAATTTCGGGGACGCTGGTACGTGGTTGGACTGGCCGGCAATGCTGTACAGAAAAAAACAGAGGGCAGTTTCACCATGTACTCAACAATCTATGAGCTCCAAGAGAATAATAGTTACAACGTTACCTCCATCTTGGTGAGGGACCAGGATCAGGGATGTCGCTACTGGATTCGGACATTCGTACCAAGTTCTCGGGCCGGTCAGTTTACTCTGGGCAACATGCACAGGTATCCCCAAGTTCAATCTTACAACGTGCAGGTGGCGACTACCGACTACAACCAATTCGCTATGGTGTTCTTCCGCAAAACAAGCGAGAACAAGCAGTATTTTAAAATCACTCTGTACGGTAGAACTAAGGAGCTGAGCCCTGAACTTAAGGAGCGGTTCACCAGATTCGCTAAGTCCCTGGGACTGAAGGATGATAATATAATCTTTTCCGTCCCCACCGATCAGTGTATCGATAATTCAGCTTGGTCACATCCCCAGTTCGAGAAAGGAGGCGGTGGATCCATGCACGGACCTAAAGCAACACTCCAGGACATCGTCCTGCATTTGGAACCACAAAACGAAATACCCGTGGACCTTTTGTGTCACGAACAGCTTTCAGATTCTGAGGAAGAGAATGATGAAATCGACGGTGTCAACCACCAGCATCTCCCCGCTAGGCGGGCAGAACCCCAGCGCCACACAATGCTGTGCATGTGTTGCAAATGCGAAGCTCGAATTGAACTCGTGGTTGAGTCCTCCGCGGACGACTTGAGGGCATTCCAGCAACTGTTCCTCAACACACTGAGCTTTGTCTGTCCTTGGTGCGCTAGTCAGCAGTGACGGACCGCCTCTCCCTCCC IgK-H6-murineScn-StrepII-GGGGS- E6.33gi|218931423|gb|ACL12326.1| E6 [HPV 33] SEQ ID NO: 60-CTCGAGACCATGGAGACCGACACGCTCTTGTTGTGGGTTCTCTTGTTGTGGGTGCCTGGGTCTACAGGCGACCACCACCATCATCACCACCTCGTTCCTAGAGGCAGCCAGGATAGTACCCAGAATCTTATCCCAGCACCATCTTTGCTCACAGTACCATTGCAACCCGACTTTCGGTCTGATCAATTTCGGGGACGCTGGTACGTGGTTGGACTGGCCGGCAATGCTGTACAGAAAAAAACAGAGGGCAGTTTCACCATGTACTCAACAATCTATGAGCTCCAAGAGAATAATAGTTACAACGTTACCTCCATCTTGGTGAGGGACCAGGATCAGGGATGTCGCTACTGGATTCGGACATTCGTACCAAGTTCTCGGGCCGGTCAGTTTACTCTGGGCAACATGCACAGGTATCCCCAAGTTCAATCTTACAACGTGCAGGTGGCGACTACCGACTACAACCAATTCGCTATGGTGTTCTTCCGCAAAACAAGCGAGAACAAGCAGTATTTTAAAATCACTCTGTACGGTAGAACTAAGGAGCTGAGCCCTGAACTTAAGGAGCGGTTCACCAGATTCGCTAAGTCCCTGGGACTGAAGGATGATAATATAATCTTTTCCGTCCCCACCGATCAGTGTATCGATAATTCAGCTTGGTCACATCCCCAGTTCGAGAAAGGAGGCGGTGGATCCATGTTCCAAGACACTGAGGAGAAGCCACGCACGCTGCACGATCTGTGCCAGGCCCTTGAGACTACCATCCATAACATCGAGCTCCAGTGTGTCGAATGCAGGAATCCTCTTCAGCGGAGCGAGGTGTACGATTTTGCCTTCGCGGACCTGACGGTGGTCTACCGGGAAGGTAACCCATTCGGGATTTGCAAGCTGTGTCTCAGATTTCTTAGTAAGATAAGTGAATACCGGCACTACAACTATTCAGTTTACGGTCACACTCTGGAACAGACCGTGAACAAACCCCTGAACGAGATCCTCATTCGATGTATCATCTGTCAGAGACCTCTCTGTCCGCGCGAAAAGAAGAGGCACGTCGACCTGAATAAGCGATTTCATAATATCTCTGGACGGTGGGCGGGGCGCTGTGCAGCCTGTTGGAGATCCCGGAGACGGGAAACAGCTCTTTGACGGACCGCCTCTCCCTCCC IgK-H6-murineScn-StrepII-GGGGS-E7.33gi|218931424|gb|ACL12327.1| E7 [HPV 33] SEQ ID NO: 61-CTCGAGACCATGGAGACCGACACGCTCTTGTTGTGGGTTCTCTTGTTGTGGGTGCCTGGGTCTACAGGCGACCACCACCATCATCACCACCTCGTTCCTAGAGGCAGCCAGGATAGTACCCAGAATCTTATCCCAGCACCATCTTTGCTCACAGTACCATTGCAACCCGACTTTCGGTCTGATCAATTTCGGGGACGCTGGTACGTGGTTGGACTGGCCGGCAATGCTGTACAGAAAAAAACAGAGGGCAGTTTCACCATGTACTCAACAATCTATGAGCTCCAAGAGAATAATAGTTACAACGTTACCTCCATCTTGGTGAGGGACCAGGATCAGGGATGTCGCTACTGGATTCGGACATTCGTACCAAGTTCTCGGGCCGGTCAGTTTACTCTGGGCAACATGCACAGGTATCCCCAAGTTCAATCTTACAACGTGCAGGTGGCGACTACCGACTACAACCAATTCGCTATGGTGTTCTTCCGCAAAACAAGCGAGAACAAGCAGTATTTTAAAATCACTCTGTACGGTAGAACTAAGGAGCTGAGCCCTGAACTTAAGGAGCGGTTCACCAGATTCGCTAAGTCCCTGGGACTGAAGGATGATAATATAATCTTTTCCGTCCCCACCGATCAGTGTATCGATAATTCAGCTTGGTCACATCCCCAGTTCGAGAAAGGAGGCGGTGGATCCATGCGGGGACATGAACCTACTCTGAAGGAGTACGTCCTGGACCTTTACCCGGAGCCGACAGATCTTTACTGTTACGAGCAATTGTCTGACTCCAGCGACGAGGATGAGGGCCTTGACAGACCTGATGGCCAGGCTCAGCCAGCTACTGCCGATTATTATATCGTTACGTGTTGTCACACCTGCAACACAACCGTAAGGTTGTGTGTGAACTCCACCGCCAGTGACTTGAGAACGATACAACAACTCCTCATGGGCACTGTCAATATCGTCTGTCCTACATGTGCTCAGCTGCTGACGGACCGCCTCTCCCTCCCIgK-H6-murineScn-StrepII-GGGGS-E6.45 gi|145968371|gb|ABP99896.1|E6 [HPV45] SEQ ID NO: 62-CTCGAGACCATGGAGACCGACACGCTCTTGTTGTGGGTTCTCTTGTTGTGGGTGCCTGGGTCTACAGGCGACCACCACCATCATCACCACCTCGTTCCTAGAGGCAGCCAGGATAGTACCCAGAATCTTATCCCAGCACCATCTTTGCTCACAGTACCATTGCAACCCGACTTTCGGTCTGATCAATTTCGGGGACGCTGGTACGTGGTTGGACTGGCCGGCAATGCTGTACAGAAAAAAACAGAGGGCAGTTTCACCATGTACTCAACAATCTATGAGCTCCAAGAGAATAATAGTTACAACGTTACCTCCATCTTGGTGAGGGACCAGGATCAGGGATGTCGCTACTGGATTCGGACATTCGTACCAAGTTCTCGGGCCGGTCAGTTTACTCTGGGCAACATGCACAGGTATCCCCAAGTTCAATCTTACAACGTGCAGGTGGCGACTACCGACTACAACCAATTCGCTATGGTGTTCTTCCGCAAAACAAGCGAGAACAAGCAGTATTTTAAAATCACTCTGTACGGTAGAACTAAGGAGCTGAGCCCTGAACTTAAGGAGCGGTTCACCAGATTCGCTAAGTCCCTGGGACTGAAGGATGATAATATAATCTTTTCCGTCCCCACCGATCAGTGTATCGATAATTCAGCTTGGTCACATCCCCAGTTCGAGAAAGGAGGCGGTGGATCCATGGCCAGGTTCGATGATCCCACCCAGCGACCCTATAAGTTGCCCGATCTCTGCACAGAACTTAACACTAGCTTGCAGGACGTAAGCATTGCATGTGTTTACTGTAAAGCTACGCTGGAGCGAACCGAGGTGTACCAATTCGCCTTCAAAGACTTGTTCATCGTGTATAGAGACTGTATCGCTTATGCCGCCTGCCACAAATGCATAGACTTTTACAGCAGGATCAGGGAATTGAGGTACTATTCCAACTCAGTCTATGGAGAAACGCTGGAGAAGATAACTAACACTGAGCTTTATAACCTCCTGATTCGCTGCCTCCGGTGTCAGAAGCCACTGAATCCTGCCGAAAAGAGACGCCATCTGAAGGACAAGCGGCGCTTTCATAGCATTGCAGGACAGTACAGAGGCCAATGTAATACTTGCTGTGACCAAGCACGCCAAGAAAGGCTCAGGAGAAGGAGAGAGACACAGGTGTGACGGACCGCCTCTCCCTCCCIgK-H6-murineScn-StrepII-GGGGS-E7.45 gi|145968372|gb|ABP99897.1|E7 [HPV 45] SEQ ID NO: 63-CTCGAGACCATGGAGACCGACACGCTCTTGTTGTGGGTTCTCTTGTTGTGGGTGCCTGGGTCTACAGGCGACCACCACCATCATCACCACCTCGTTCCTAGAGGCAGCCAGGATAGTACCCAGAATCTTATCCCAGCACCATCTTTGCTCACAGTACCATTGCAACCCGACTTTCGGTCTGATCAATTTCGGGGACGCTGGTACGTGGTTGGACTGGCCGGCAATGCTGTACAGAAAAAAACAGAGGGCAGTTTCACCATGTACTCAACAATCTATGAGCTCCAAGAGAATAATAGTTACAACGTTACCTCCATCTTGGTGAGGGACCAGGATCAGGGATGTCGCTACTGGATTCGGACATTCGTACCAAGTTCTCGGGCCGGTCAGTTTACTCTGGGCAACATGCACAGGTATCCCCAAGTTCAATCTTACAACGTGCAGGTGGCGACTACCGACTACAACCAATTCGCTATGGTGTTCTTCCGCAAAACAAGCGAGAACAAGCAGTATTTTAAAATCACTCTGTACGGTAGAACTAAGGAGCTGAGCCCTGAACTTAAGGAGCGGTTCACCAGATTCGCTAAGTCCCTGGGACTGAAGGATGATAATATAATCTTTTCCGTCCCCACCGATCAGTGTATCGATAATTCAGCTTGGTCACATCCCCAGTTCGAGAAAGGAGGCGGTGGATCCATGCACGGCCCACAGGCAACCCTGCAAGAGATCGTGCTGCATCTCGAACCACAGAATGAATTGGACCCTGTGGATCTGCTGTGTTACGAGCAGCTCTCTGAAAGCGAAGAGGAGAATGACGAGGCCGACGGCGTGTCTCATGCACAGCTGCCTGCTCGCCGGGCCGAACCTCAGCGACACAAAATTCTGTGCGTGTGCTGCAAATGCGACGGCCGCATAGAGCTGACGGTAGAATCATCAGCCGACGATCTGCGAACTCTTCAACAACTCTTCCTGAGCACGCTCAGCTTCGTGTGTCCTTGGTGTGCTACAAATCAGTGACGGACCGCCTCTCCCTCCC IgK-H6-murineScn-StrepII-GGGGS-E6.31gi|l48727550|gb|ABR08438.1| E6 protein [HPV31] SEQ ID NO: 64-CTCGAGACCATGGAGACCGACACGCTCTTGTTGTGGGTTCTCTTGTTGTGGGTGCCTGGGTCTACAGGCGACCACCACCATCATCACCACCTCGTTCCTAGAGGCAGCCAGGATAGTACCCAGAATCTTATCCCAGCACCATCTTTGCTCACAGTACCATTGCAACCCGACTTTCGGTCTGATCAATTTCGGGGACGCTGGTACGTGGTTGGACTGGCCGGCAATGCTGTACAGAAAAAAACAGAGGGCAGTTTCACCATGTACTCAACAATCTATGAGCTCCAAGAGAATAATAGTTACAACGTTACCTCCATCTTGGTGAGGGACCAGGATCAGGGATGTCGCTACTGGATTCGGACATTCGTACCAAGTTCTCGGGCCGGTCAGTTTACTCTGGGCAACATGCACAGGTATCCCCAAGTTCAATCTTACAACGTGCAGGTGGCGACTACCGACTACAACCAATTCGCTATGGTGTTCTTCCGCAAAACAAGCGAGAACAAGCAGTATTTTAAAATCACTCTGTACGGTAGAACTAAGGAGCTGAGCCCTGAACTTAAGGAGCGGTTCACCAGATTCGCTAAGTCCCTGGGACTGAAGGATGATAATATAATCTTTTCCGTCCCCACCGATCAGTGTATCGATAATTCAGCTTGGTCACATCCCCAGTTCGAGAAAGGAGGCGGTGGATCCATGTTCAAAAACCCGGCTGAGAGACCGCGGAAGTTGCACGAGCTCTCATCCGCGCTGGAAATACCTTATGATGAGCTTCGCTTGAATTGTGTGTACTGCAAAGGCCAGCTCACTGAGACCGAAGTACTTGATTTTGCCTTTACTGACCTGACAATCGTCTATAGAGACGACACTCCACACGGGGTCTGTACAAAATGTCTGCGGTTTTATAGTAAAGTGAGCGAATTCCGGTGGTATCGCTATTCAGTGTATGGAACCACATTGGAGAAACTCACTAACAAAGGTATCTGTGACCTGCTGATCAGGTGCATAACTTGTCAGAGGCCGCTCTGCCCCGAGGAGAAGCAGCGCCACCTGGATAAGAAGAAGAGATTCCACAACATTGGAGGCAGATGGACAGGCCGGTGCATTGCTTGTTGGCGCAGGCCAAGAACCGAGACCCAAGTTTGACGGACCGCCTCTCCCTCCC IgK-H6-murineScn-StrepII-GGGGS-E7.31gi|338969947|gb|AEJ33624.1| E7, partial [HPV31] SEQ ID NO: 65-CTCGAGACCATGGAGACCGACACGCTCTTGTTGTGGGTTCTCTTGTTGTGGGTGCCTGGGTCTACAGGCGACCACCACCATCATCACCACCTCGTTCCTAGAGGCAGCCAGGATAGTACCCAGAATCTTATCCCAGCACCATCTTTGCTCACAGTACCATTGCAACCCGACTTTCGGTCTGATCAATTTCGGGGACGCTGGTACGTGGTTGGACTGGCCGGCAATGCTGTACAGAAAAAAACAGAGGGCAGTTTCACCATGTACTCAACAATCTATGAGCTCCAAGAGAATAATAGTTACAACGTTACCTCCATCTTGGTGAGGGACCAGGATCAGGGATGTCGCTACTGGATTCGGACATTCGTACCAAGTTCTCGGGCCGGTCAGTTTACTCTGGGCAACATGCACAGGTATCCCCAAGTTCAATCTTACAACGTGCAGGTGGCGACTACCGACTACAACCAATTCGCTATGGTGTTCTTCCGCAAAACAAGCGAGAACAAGCAGTATTTTAAAATCACTCTGTACGGTAGAACTAAGGAGCTGAGCCCTGAACTTAAGGAGCGGTTCACCAGATTCGCTAAGTCCCTGGGACTGAAGGATGATAATATAATCTTTTCCGTCCCCACCGATCAGTGTATCGATAATTCAGCTTGGTCACATCCCCAGTTCGAGAAAGGAGGCGGTGGATCCATGCGGGGTGAGACACCAACTCTTCAGGATTATGTTCTGGATCTGCAGCCAGAGGCCACAGATCTGCACTGTTACGAGCAATTGCCTGATTCCAGCGACGAGGAGGATGTCATCGATAGCCCTGCTGGGCAGGCCAAGCCAGACACTTCAAATTACAACATTGTAACGTTTTGTTGTCAGTGCGAATCCACCCTCAGGCTTTGCGTCCAGAGCACTCAGGTTGACATTCGAATACTCCAGGAGCTGTTGATGGGGAGCTTTGGAATCGTGTGCCCAAATTGTAGTACACGACTGTGACGGACCGCCTCTCCCTCCCIgK-H6-murineScn-StrepII-GGGGS-E6.58 gi|425892429|gb|AFY09749.1|E6 [HPV 58] SEQ ID NO: 66-CTCGAGACCATGGAGACCGACACGCTCTTGTTGTGGGTTCTCTTGTTGTGGGTGCCTGGGTCTACAGGCGACCACCACCATCATCACCACCTCGTTCCTAGAGGCAGCCAGGATAGTACCCAGAATCTTATCCCAGCACCATCTTTGCTCACAGTACCATTGCAACCCGACTTTCGGTCTGATCAATTTCGGGGACGCTGGTACGTGGTTGGACTGGCCGGCAATGCTGTACAGAAAAAAACAGAGGGCAGTTTCACCATGTACTCAACAATCTATGAGCTCCAAGAGAATAATAGTTACAACGTTACCTCCATCTTGGTGAGGGACCAGGATCAGGGATGTCGCTACTGGATTCGGACATTCGTACCAAGTTCTCGGGCCGGTCAGTTTACTCTGGGCAACATGCACAGGTATCCCCAAGTTCAATCTTACAACGTGCAGGTGGCGACTACCGACTACAACCAATTCGCTATGGTGTTCTTCCGCAAAACAAGCGAGAACAAGCAGTATTTTAAAATCACTCTGTACGGTAGAACTAAGGAGCTGAGCCCTGAACTTAAGGAGCGGTTCACCAGATTCGCTAAGTCCCTGGGACTGAAGGATGATAATATAATCTTTTCCGTCCCCACCGATCAGTGTATCGATAATTCAGCTTGGTCACATCCCCAGTTCGAGAAAGGAGGCGGTGGATCCATGTTTCAGGACGCTGAGGAGAAGCCCAGAACTCTGCACGATCTGTGTCAGGCCTTGGAGACGTCTGTGCATAAAATTGAGCTTAAATGTGTCGAATGTAAGAAGACACTCCAGCGCAGCGAAGTTTATGACTTCGTGTTCGCGGATCTGAGAATCGTGTATCGGGACGGCAACCCTTTTGCTGTTTGCAAGGTTTGCCTTAGGCTCCTGTCCAAAATTAGCGAGTACCGCCACTATAACTACTCTCTCTACGGTGATACTCTCGAGCAAACACTGAAGAAGTGCTTGAACGAGATCCTGATTAGATGCATCATTTGTCAAAGGCCACTTTGTCCACAGGAGAAGAAGAGGCACGTGGACCTGAATAAGCGCTTTCATAACATCTCTGGCAGATGGACAGGCCGATGCGCTGTATGTTGGCGCCCACGGAGAAGGCAAACCCAGGTGTGACGGACCGCCTCTCCCTCCC IgK-H6-murineScn-StrepII-GGGGS-E7.58gi|414090989|gb|AFW98384.1| E7 [HPV 58] SEQ ID NO: 67-CTCGAGACCATGGAGACCGACACGCTCTTGTTGTGGGTTCTCTTGTTGTGGGTGCCTGGGTCTACAGGCGACCACCACCATCATCACCACCTCGTTCCTAGAGGCAGCCAGGATAGTACCCAGAATCTTATCCCAGCACCATCTTTGCTCACAGTACCATTGCAACCCGACTTTCGGTCTGATCAATTTCGGGGACGCTGGTACGTGGTTGGACTGGCCGGCAATGCTGTACAGAAAAAAACAGAGGGCAGTTTCACCATGTACTCAACAATCTATGAGCTCCAAGAGAATAATAGTTACAACGTTACCTCCATCTTGGTGAGGGACCAGGATCAGGGATGTCGCTACTGGATTCGGACATTCGTACCAAGTTCTCGGGCCGGTCAGTTTACTCTGGGCAACATGCACAGGTATCCCCAAGTTCAATCTTACAACGTGCAGGTGGCGACTACCGACTACAACCAATTCGCTATGGTGTTCTTCCGCAAAACAAGCGAGAACAAGCAGTATTTTAAAATCACTCTGTACGGTAGAACTAAGGAGCTGAGCCCTGAACTTAAGGAGCGGTTCACCAGATTCGCTAAGTCCCTGGGACTGAAGGATGATAATATAATCTTTTCCGTCCCCACCGATCAGTGTATCGATAATTCAGCTTGGTCACATCCCCAGTTCGAGAAAGGAGGCGGTGGATCCATGCGGGGGAATAACCCCACCCTGCGCGAGTACATTCTTGACCTGCACCCAGAGCCTACGGATCTGTTTTGTTACGAACAACTGTGCGACTCCTCCGACGAGGATGAGATCGGGCTGGATGGCCCAGACGGGCAGGCACAGCCTGCTACAGCTAACTACTATATTGTGACATGTTGCTACACATGCGGAACGACGGTCAGACTGTGCATTAATAGCACTGCCACAGACGTGCGGACCCTGCAGCAACTGCTCATGGGGACCTGCACTATTGTGTGTCCTTCATGTGCGCAGCAATGACGGACCGCCTCTCCCTCCCHuman Scn construct for downstream viral fusions:IgK-sFLAG-H6-GGS-humanScn-GGS-ENLYFQ-GG-Adv2E3/19K SEQ ID NO: 68-METDTLLLWVLLLWVPGSTGDYKDEHHHHHHGGSQDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGSQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDGGGSENLYFQGGAKKVEFKEPACNVTFKSEANECTTLIKCTTEHEKLIIRHKDKIGKYAVYAIWQPGDTNDYNVTVFQGENRKTFMYKFPFYEMCDITMYMSKQ YKLWIgK-sFLAG-H6-GGS-humanScn-GGS-ENLYFQ-GG-SF162gp120 SEQ ID NO: 69-METDTLLLWVLLLWVPGSTGDYKDEHHHHHHGGSQDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGSQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDGGGSENLYFQGGWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEIVLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLHCTNLKNATNTKSSNWKEMDRGEIKNCSFKVTTSIRNKMQKEYALFYKLDVVPIDNDNTSYKLINCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNDKKFNGSGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEGVVIRSENFTDNAKTIIVQLKESVEINCTRPNNNTRKSITIGPGRAFYATGDIIGDIRQAHCNISGEKWNNTLKQIVTKLQAQFGNKTIVFKQSSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWNNTIGPNNTNGTITLPCRIKQIINRWQEVGKAMYAPPIRGQIRCSSNITGLLLTRDGGKEISNTTEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQQGLNDIFEAQKIEW HEIgK-sFLAG-H6-GGS-humanScn-GGS-ENLYFQ-GG-QH0692gp120 SEQ ID NO: 70-METDTLLLWVLLLWVPGSTGDYKDEHHHHHHGGSQDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGSQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDGGGSENLYFQGGWVTVYYGVPVWKEATTTLFCASDAKAYETEKHNVWATHACVPTDPNPQEVVLGNVTENFNMWKNNMVEQMHEDIISLWDESLKPCVKLTPLCVTLNCTDEVKTSYANKTSNETYKTSNETFGEIKNCSFSVPTGIKDKVQNVYALFYKLDVIPIDDNNNSSKNNNGSYSSYRLINCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSENFTNNAKTIIVHLKKSVEINCTRPGNNTRKSIHIGPGRAFYATGDIIGDIRQAHCNLSSVQWNDTLKQIVIKLGEQFGTNKTIAFNQSSGGDPEIVMHSFNCGGEFFYCNTTQLFNSTWEFHGNWTRSNFTESNSTTITLPCRIKQIVNMWQEVGKAMYAPPIRGQIRCSSNITGLLLTRDGGVNGTRETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQGLNDIFEAQKIEWHEHuman Scn construct for downstream peptide fusions:IgK-sFLAG-H6-GGS-humanScn-GGS-ENLYFQ-GG-ITPR-1 SEQ ID NO: 71-METDTLLLWVLLLWVPGSTGDYKDEHHHHHHGGSQDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGSQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDGGGSENLYFQGGSKCRVFNTTERDEQGSKVNDFFQQTEDLYNEMKWQK IgK-sFLAG-H6-GGS-humanScn-GGS-ENLYFQ-GG-ITPR-2 SEQ ID NO: 72-METDTLLLWVLLLWVPGSTGDYKDEHHHHHHGGSQDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGSQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDGGGSENLYFQGGPPHELTEEEKQQILHSEEFLSFFDHSTRIVERALSE IdK-sFLAG-H6-GGS-humanScn-GGS-ENLYFQ-GG-ITPR-3 SEQ ID NO: 73-METDTLLLWVLLLWVPGSTGDYKDEHHHHHHGGSQDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGSQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDGGGSENLYFQGGPPPRCISTNKCTAPEVENAIRVPGNRSFFSLTEIVR IgK-sFLAG-H6-GGS-humanScn-GGS-ENLYFQ-GG-ITPR-4 SEQ ID NO: 74-METDTLLLWVLLLWVPGSTGDYKDEHHHHHHGGSQDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGSQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDGGGSENLYFQGGTERDEQGSKINDFFLRSEDLFNEMNWQKKL RAQPVLIgK-sFLAG-H6-GGS-humanScn-GGS-ENLYFQ-GG-ITPR-5 SEQ ID NO: 75-METDTLLLWVLLLWVPGSTGDYKDEHHHHHHGGSQDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGSQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDGGGSENLYFQGGLTEETKHRLFTTTEQDEQGSKVSDFFDQSSFLHNEM IgK-sFLAG-H6-GGS-humanScn-GGS-ENLYFQ-GG-ITPR-8 SEQ ID NO: 76-METDTLLLWVLLLWVPGSTGDYKDEHHHHHHGGSQDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGSQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDGGGSENLYFQGGGAQPPFDAQSPLDSQPQPSGQPWNFHASTSWYWRQS Human Scn construct for downstream HMOX1 fusions:IgK-5FLAG-H6-GGS-humanScn-GGS-ENLYFQ-GG-human HMOX1 SEQ ID NO: 77-METDTLLLWVLLLWVPGSTGDYKDEHHHHHHGGSQDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGSQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDGGGSENLYFQGGMERPQPDSMPQDLSEALKEATKEVHTQAENAEFMRNFQKGQVTRDGFKLVMASLYHIYVALEEEIERNKESPVFAPVYFPEELHRKAALEQDLAFWYGPRWQEVIPYTPAMQRYVKRLHEVGRTEPELLVAHAYTRYLGDLSGGQVLKKIAQKALDLPSSGEGLAFFTFPNIASATKFKQLYRSRMNSLEMTPAVRQRVIEEAKTAFLLNIQLFEELQELLTHDTKDQSPSRAPGLRQRASNKVQDSAPVETPRGKPPLNTRSQAIgK-sFLAG-H6-GGS-humanScn-GGS-ENLYFQ-GG-murineHMOX1 SEQ ID NO: 78-METDTLLLWVLLLWVPGSTGDYKDEHHHHHHGGSQDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGSQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDGGGSENLYFQGGMERPQPDSMPQDLSEALKEATKEVHIQAENAEFMKNFQKGQVSREGFKLVMASLYHIYTALEEEIERNKQNPVYAPLYFPEELHRRAALEQDMAFWYGPHWQEIIPCTPATQHYVKRLHEVGRTHPELLVAHAYTRYLGDLSGGQVLKKIAQKAMALPSSGEGLAFFTFPNIDSPTKFKQLYRARMNTLEMTPEVKHRVTEEAKTAFLLNIELFEELQVMLTEEHKDQSPSQMASLRQRPASLVQDTAPAETPRGKPQISTSSSQ*humanScn-humanHMOX1 (Second Generation) SEQ ID NO: 79-MPLGLLWLGLALLGALHAQAQDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGSQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDGGGSENLYFQGGGMERPQPDSMPQDLSEALKEATKEVHTQAENAEFMRNFQKGQVTRDGFKLVMASLYHIYVALEEEIERNKESPVFAPVYFPEELHRKAALEQDLAFWYGPRWQEVIPYTPAMQRYVKRLHEVGRTEPELLVAHAYTRYLGDLSGGQVLKKIAQKALDLPSSGEGLAFFTFPNIASATKFKQLYRSRMNSLEMTPAVRQRVIEEAKTAFLLNIQLFEELQELLTHDTKDQSPSRAPGLRQRASNKVQDSAPVETPRGKPPLNTRSQAGGLVPRGSHHHHH HhumanScn-cTHAP4 SEQ ID NO: 80-MPLGLLWLGLALLGALHAQAQDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGSQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDGGGSENLYFQGGGPPKMNPVVEPLSWMLGTWLSDPPGAGTYPTLQPFQYLEEVHISHVGQPMLNFSFNSFHPDTRKPMHRECGFIRLKPDTNKVAFVSAQNTGVVEVEEGEVNGQELCIASHSIARISFAKEPHVEQITRKFRLNSEGKLEQTVSMATTTQPMTQHLHVTYKKVTPGGLVPRGSHHHHHHHuman Scn construct for downstream multimer fusions:IgK-sFLAG-H6-GGS-humanScn-GGS-ENLYFQ-GG-heptamer SEQ ID NO: 81-METDTLLLWVLLLWVPGSTGDYKDEHHHHHHGGSQDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGSQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDGGGSENLYFQGGGRSAGAHAGWETPEGCEQVLTGKRLMQCLPNPEDVKMALEVYKLSLEIEQLELQRDSARQSTLDKELVPRGSIgK-sFLAG-H6-GGS-humanScn-GGS-ENLYFQ-GG-CD80heptamer SEQ ID NO: 82-METDTLLLWVLLLWVPGSTGDYKDEHHHHHHGGSQDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGSQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDGGGSENLYFQGGGIIQVNKTVKEVAVLSCDYNISTTELMKVRIYWQKDDEVVLAVTSGQTKVWSKYENRTFADFTNNLSIVIMALRLSDNGKYTCIVQKTEKRSYKVKHMTSVMLLVRADFPVPSITDLGNPSHDIKRIMCSTSGGFPKPHLSWWENEEELNAANTTVSQDPDTELYTISSELDFNITSNHSFVCLVKYGDLTVSQIFNWQKSVEPHPPNNSAWSHPQFEKGGSLVPRGSGSAGAHAGWETPEGCEQVLTGKRLMQCLPNPEDVKMALXVYKLSLEIEQLELQRDSARQSTLDKELVPRGS IgK-sFLAG-H6-GGS-humanScn-GGS-ENLYFQ-GG-trimerSEQ ID NO: 83-METDTLLLWVLLLWVPGSTGDYKDEHHHHHHGGSQDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGSQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDGGGSENLYFQGGGRNLVTAFSNMDDMLQKAHLVIEGTFIYLRDSTEFFIRVRDGWKKLQLGELIPIPAIgK-sFLAG-H6-GGS-humanScn-GGS-ENLYFQ-GG-CD80trimer SEQ ID NO: 84-METDTLLLWVLLLWVPGSTGDYKDEHHHHHHGGSQDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGSQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDGGGSENLYFQGGGIIQVNKTVKEVAVLSCDYNISTTELMKVRIYWQKDDEVVLAVTSGQTKVWSKYENRTFADFTNNLSIVIMALRLSDNGKYTCIVQKTEKRSYKVKHMTSVMLLVRADFPVPSITDLGNPSHDIKRIMCSTSGGFPKPHLSWWENEEELNAANTTVSQDPDTELYTISSELDFNITSNHSFVCLVKYGDLTVSQIFNWQKSVEPHPPNNSAWSHPQFEKGGSLVPRGSGNLVTAFSNMDDMLQKAHLVIEGTFIYLRDSTEFFIRVRDGWKKLQLGELIPIPAIgK-sFLAG-H6-GGS-humanScn-GGS-ENLYFQ-GG-pentamer SEQ ID NO: 85-METDTLLLWVLLLWVPGSTGDYKDEHHHHHHGGSQDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGSQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDGGGSENLYFQGGGRSSNAKWDQWSSDWQTWNAKWDQWSNDWNAWRSDWQAWKDDWARWNQRWDNWATHuman Scn construct for downstream subdomain fusions:IgK-sFLAG-H6-GGS-humanScn-GGS-ENLYFQ-GG-ROR1Kringle SEQ ID NO: 86-METDTLLLWVLLLWVPGSTGDYKDEHHHHHHGGSQDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGSQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDGGGSENLYFQGGCYNSTGVDYRGTVSVTKSGRQCQPWNSQYPHTHTFTALRFPELNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPACIgK-sFLAG-H6-GGS-humanScn-GGS-ENLYFQ-GG-CTLA4 SEQ ID NO: 87-METDTLLLWVLLLWVPGSTGDYKDEHHHHHHGGSQDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGSQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDGGGSENLYFQGGMHVAQPAVVLASSRGVASFVCEYGSSGNAAEVRVTVLRQAGSQMTEVCAATYTVEDELAFLDDSTCTGTSSGNKVNLTIQGLRAMDTGLYICKVELMYPPPYYVGMGNGTQIYVIDPEPCHuman Scn construct for downstream knottin fusions:IgK-sFLAG-H6-GGS-humanScn-GGS-ENLYFQ-GG-Imperatoxin SEQ ID NO: 88-CTCGAGACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACTACAAGGACGAGCATCACCATCATCACCATGGTGGAAGCCAGGACTCCACCTCAGACCTGATCCCAGCCCCACCTCTGAGCAAGGTCCCTCTGCAGCAGAACTTCCAGGACAACCAATTCCAGGGGAAGTGGTATGTGGTAGGCCTGGCAGGGAATGCAATTCTCAGAGAAGACAAAGACCCGCAAAAGATGTATGCCACCATCTATGAGCTGAAAGAAGACAAGAGCTACAATGTCACCTCCGTCCTGTTTAGGAAAAAGAAGTGTGACTACTGGATCAGGACTTTTGTTCCAGGTTGCCAGCCCGGCGAGTTCACGCTGGGCAACATTAAGAGTTACCCTGGATTAACGAGTTACCTCGTCCGAGTGGTGAGCACCAACTACAACCAGCATGCTATGGTGTTCTTCAAGAAAGTTTCTCAAAACAGGGAGTACTTCAAGATCACCCTCTACGGGAGAACCAAGGAGCTGACTTCGGAACTAAAGGAGAACTTCATCCGCTTCTCCAAATCTCTGGGCCTCCCTGAAAACCACATCGTCTTCCCTGTCCCAATCGACCAGTGTATCGACGGCGGAGGTAGCGAAAACCTGTATTTTCAGGGAGGCGACTGCCTGCCCCACCTGAGGAGGTGCAGGGCCGACAACGACTGCTGCGGCAGGAGGTGCAGGAGGAGGGGCACCAACGCCGAGAGGAGGTGCAGGTAAGCTAAGGATCC IgK SP: Murine IgK light chain signal peptide:SEQ ID NO: 89- METDTLLLWVLLLWVPGSTG SEQ ID NO: 90-ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTsFLAG: Shortened FLAG epitope: SEQ ID NO: 91- DYKDE SEQ ID NO: 92-GACTACAAGGACGAG HIS: 6xhistidine tag SEQ ID NO: 93- HHHHHHSEQ ID NO: 94- CATCATCATCATCATCATTEV: Tobacco Etch Virus Protease recognition site: SEQ ID NO: 95- ENLYFQSEQ ID NO: 96- GAGAATTTATATTTTCAG SEQ ID NO: 97-Furin - furin cleavage site with BamHI site (GGATCC): SEQ ID NO: 99-RARYKRGS SEQ ID NO: 100 CGGGCCCGGTATAAACGGGGATCC

What is claimed is:
 1. A method for producing a fusion protein, themethod comprising expressing, in a cell, a fusion protein, the fusionprotein comprising human siderocalin and trastuzumab or a fragmentthereof.
 2. A method of claim 1, wherein the expressing utilizes theparental cloning construct of SEQ ID NO:
 37. 3. A method of claim 1,wherein the fusion protein comprises trastuzumab.
 4. A method of claim1, wherein the fusion protein comprises a fragment of trastuzumab.
 5. Amethod of claim 4, wherein the fragment is a Fab fragment.
 6. A methodof claim 1, further comprising associating the human siderocalin with aradionuclide.
 7. A method of claim 5, wherein the radionuclide isselected from Sc-47, Ga-67, Y-90, Ag-111, In-111, Sm-153, Tb-166,Lu-177, Bi-213, Ac-225, Cu-64, Cu-67, Pd-109, Ag-111, Re-186, Re-188,Pt-197, Bi-212, Bi-213, Pb-212 or Ra-223.
 8. A fusion protein producedby the method of claim
 1. 9. A fusion protein of claim 7, wherein thefusion protein comprises trastuzumab.
 10. A fusion protein of claim 7,wherein the fusion protein comprises a fragment of trastuzumab.
 11. Afusion protein of claim 10, wherein the fragment is a Fab fragment. 12.A fusion protein of claim 7, wherein the human siderocalin is associatedwith a radionuclide.
 13. A fusion protein of claim 12, wherein theradionuclide is selected from Sc-47, Ga-67, Y-90, Ag-111, In-111,Sm-153, Tb-166, Lu-177, Bi-213, Ac-225, Cu-64, Cu-67, Pd-109, Ag-111,Re-186, Re-188, Pt-197, Bi-212, Bi-213, Pb-212 or Ra-223.
 14. A methodfor producing a fusion protein, the method comprising expressing, in acell, a fusion protein, the fusion protein comprising human siderocalinand infliximab or a fragment thereof, adalimumab or a fragment thereof,OKT3 or a fragment thereof, or Fc.
 15. A method of claim 14, wherein theexpressing utilizes the parental cloning construct of SEQ ID NO:
 37. 16.A method of claim 14, wherein the fusion protein comprises infliximab,adalimumab, OKT3, or Fc.
 17. A method of claim 14, wherein the fusionprotein comprises a fragment of infliximab, a fragment of adalimumab, ora fragment of OKT3.
 18. A method of claim 17, wherein the fragment is aFab fragment.
 19. A method of claim 14, further comprising associatingthe human siderocalin with a radionuclide.
 20. A method of claim 19,wherein the radionuclide is selected from Sc-47, Ga-67, Y-90, Ag-111,In-111, Sm-153, Tb-166, Lu-177, Bi-213, Ac-225, Cu-64, Cu-67, Pd-109,Ag-111, Re-186, Re-188, Pt-197, Bi-212, Bi-213, Pb-212 or Ra-223.